Immunogenic compositions for use in pneumococcal vaccines

ABSTRACT

An object of the present invention is to provide immunogenic compositions for protection against S. pneumoniae, in particular against S. pneumoniae serogroup 10A and 39, while limiting the number of conjugates. The present invention therefore relates to new immunogenic compositions for use in pneumococcal vaccines and to vaccination of human subjects, in particular infants and elderly, against pneumoccocal infections using said immunogenic compositions.

FIELD OF THE INVENTION

The present invention relates to new immunogenic compositions for use inpneumococcal vaccines. Immunogenic compositions of the present inventionwill typically comprise conjugated capsular saccharide antigens(glycoconjugates), wherein the saccharides are derived from serotypes ofStreptococus pneumoniae. An object of the present invention is toprovide immunogenic compositions for protection against S. pneumoniaeserogroups 10A and 39.

BACKGROUND OF THE INVENTION

Infections caused by pneumococci are a major cause of morbidity andmortality all over the world. Pneumonia, febrile bacteraemia andmeningitis are the most common manifestations of invasive pneumococcaldisease, whereas bacterial spread within the respiratory tract mayresult in middle-ear infection, sinusitis or recurrent bronchitis.Compared with invasive disease, the non-invasive manifestations areusually less severe, but considerably more common.

The etiological agent of pneumococcal diseases, Streptococcus pneumoniae(pneumococcus), is a Gram-positive encapsulated coccus, surrounded by apolysaccharide capsule. The capsular polysaccharides (CPs) are keyantigenic determinants of this bacterium and protective immune responsesto pneumococci in humans are typically directed against the capsularpolysaccharides. Differences in the composition of this capsule permitserological differentiation between about 91 capsular types, some ofwhich are frequently associated with pneumococcal disease, othersrarely. Invasive pneumococcal infections include pneumonia, meningitisand febrile bacteremia; among the common non-invasive manifestations areotitis media, sinusitis and bronchitis.

Protection against a high number of serotypes, while limiting the numberof conjugates in the composition, maybe very difficult to obtain despiteof the significant value. An object of the present invention is toprovide immunogenic compositions for appropriate protection against S.pneumoniae, in particular against S. pneumoniae serogroup 10A and 39,while limiting the number of conjugates.

SUMMARY OF THE INVENTION

The present invention relates to an immunogenic composition comprisingat least one glycoconjugate from S. pneumoniae serotype 39.

In one aspect the present invention further relates to an immunogeniccomposition comprising at least one glycoconjugate from S. pneumoniaeserotype 39 for use in a method of immunizing a subject againstinfection by S. pneumoniae serotype 10A. Preferably said compositiondoes not comprise capsular saccharide from S. pneumoniae serotypes 10A.

The present invention also relates to an immunogenic compositioncomprising at least one glycoconjugate from S. pneumoniae serotype 10Afor use in a method of immunizing a subject against infection by S.pneumoniae serotype 39. Preferably said composition does not comprisecapsular saccharide from S. pneumoniae serotypes 39. In one aspect thepresent invention relates to the use of an immunogenic compositioncomprising at least one glycoconjugate from S. pneumoniae serotype 10Afor the manufacture of a medicament for immunizing a subject againstinfection by S. pneumoniae serotype 39. Preferably said composition doesnot comprise capsular saccharide from S. pneumoniae serotypes 39.

In one aspect the present invention relates to the use of an immunogeniccomposition comprising at least one glycoconjugate from S. pneumoniaeserotype 39 for the manufacture of a medicament for immunizing a subjectagainst infection by S. pneumoniae serotype 10A. Preferably saidcomposition does not comprise capsular saccharide from S. pneumoniaeserotypes 10A.

In one aspect, the above immunogenic compositions further comprise atleast one glycoconjugate from S. pneumoniae serotypes 4, 6B, 9V, 14,18C, 19F and/or 23F.

In an aspect the above immunogenic compositions further comprise atleast one glycoconjugate from S. pneumoniae serotype 1, 5 and/or 7F.

In an aspect the above immunogenic compositions further comprise atleast one glycoconjugate from S. pneumoniae serotype 6A and/or 19A.

In an aspect the above immunogenic compositions further comprise atleast one glycoconjugate from S. pneumoniae serotype 3, 15B, 22F, 33F,12F, 11A and/or 8.

In a further aspect the above immunogenic compositions further compriseat least one glycoconjugate from S. pneumoniae serotype 2, 9N, 15C, 17Fand/or 20.

In a further aspect the immunogenic compositions is a 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-valent pneumococcalconjugate composition.

In a further aspect the glycoconjugates of the immunogenic compositionsare individually conjugated to CRM197.

In on aspect, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5,6B, 7F, 9V, 14 and/or 23F of the immunogenic compositions areindividually conjugated to PD, and if present, the glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT and the glycoconjugatefrom S. pneumoniae serotype 19F is conjugated to DT.

In on aspect, the glycoconjugates are prepared using CDAP chemistry orby reductive amination chemistry.

The immunogenic composition may further comprise antigens from otherpathogens, and/or at least one adjuvant such as aluminum phosphate,aluminum sulphate or aluminum hydroxide.

In an aspect the immunogenic compositions are able to elicit IgGantibodies in human which are capable of binding S. pneumoniae serotypes10A and/or 39 polysaccharide at a concentration of at least 0.35 μg/mlas determined by ELISA assay.

In an aspect the immunogenic compositions are able to elicit a titer ofat least 1:8 against S. pneumoniae serotype 10A and/or 39 in at least50% of the subjects as determined by in vitro opsonophagocytic killingassay (OPA).

In an aspect the immunogenic compositions are able to significantlyincrease the proportion of responders against S. pneumoniae serotype 10Aand/or 39 as compared to the pre-immunized population.

In an aspect the immunogenic compositions are able to significantlyincrease the OPA titers of human subjects against S. pneumoniae serotype10A and/or 39 as compared to the pre-immunized population.

In an aspect the immunogenic compositions are for use in a method ofimmunizing a subject against infection by S. pneumoniae serotype 10Aand/or 39.

In an aspect the immunogenic compositions are for use in a method forpreventing, treating or ameliorating an infection, disease or conditioncaused by S. pneumoniae serotypes 10A and/or 39 in a subject, for use toprevent serotypes 10A and/or 39 S. pneumoniae infection in a subject orfor use in a method to protect or treat a human susceptible to S.pneumoniae serotypes 10A and/or 39 infection, by means of administeringsaid immunogenic compositions via a systemic or mucosal route.

In one aspect the present invention relates to the use of theimmunogenic composition disclosed in the present document for themanufacture of a medicament for preventing, treating or ameliorating aninfection, disease or condition caused by S. pneumoniae serotypes 10Aand/or 39 in a subject

In one aspect the present invention relates to the use of theimmunogenic composition disclosed in the present document for use toprevent serotypes 10A and/or 39 S. pneumoniae infection in a subject orfor use in a method to protect or treat a human susceptible to S.pneumoniae serotypes 10A and/or 39 infection, by means of administeringsaid immunogenic compositions via a systemic or mucosal route.

In an aspect the invention relates to a method of preventing, treatingor ameliorating an infection, disease or condition associated with S.pneumoniae serotypes 10A and/or 39 in a subject, comprisingadministering to the subject an immunologically effective amount of theimmunogenic composition of the invention.

In an aspect the invention relates to a method of preventing aninfection by S. pneumoniae serotypes 10A and/or 39 in a subject,comprising administering to the subject an immunologically effectiveamount of the immunogenic composition of the invention.

The invention further relates to a kit comprising an immunogeniccomposition disclosed herein and an information leaflet, wherein saidinformation leaflet mentions the ability of the composition to elicitfunctional antibodies against S. pneumoniae serotypes 10A and/or 39 andprocess for producing said kit.

It has been surprisingly found that serotype 10A polysaccharideconjugate beyond eliciting functional reactive antibodies to serogroup10A, can additionally elicit functional, cross-reactive antibodies toserotype 39.

It has also been surprisingly found that serotype 39 polysaccharideconjugate beyond eliciting functional reactive antibodies to serogroup39, can additionally elicit functional, cross-reactive antibodies toserotype 10A.

FIGURES

FIG. 1: Cross-reactivity of S. pneumoniae 10A specific mAb with S.pneumoniae 39. Bacteria by FACS

FIG. 2: Cross-reactivity of S. pneumoniae 10A specific mAb with S.pneumoniae 39 Bacteria by multiplex urinary antigen detection (UAD)assay (B)

FIG. 3: S. pneumoniae 10A mAb reacts with S. pneumoniae 10A and 39capsular polysaccharides (CPs) in UAD assay but not with other CPs of S.pneumoniae serogroup 10: 10B, 100 and 10A FIG. 4: OPA Killing Titers ofS. pn. 10A, 11A and 39 strains with S. pn. 10A Specific mAbs

FIG. 5: Inhibition of OPA Killing of S. pn. 10A Bacteria with S. pn. 10AmAbs with S.pn. 39 and 10 Capsular Polysaccharides.

FIG. 6: Inhibition of OPA Killing of S. pn. 39 Bacteria by S. pn. 10AmAbs with S.pn. 39 and serogroup 10 Capsular Polysaccharides.

FIG. 7: S. pn. 10A and 39 bacteria are killed by 23v S. pn.polysaccharide vaccine immune human sera (n=39).

FIG. 8: OPA killing of S. pn. 10A bacteria with 23v CPs vaccine immunehuman sera in the presence or absence of homologous 10A and heterologousCPs.

FIG. 9: S. pn. 39 CP-CRM₁₉₇ and S. pn. 39 CP-TT conjugates sera canmediate killing of S. pn. 39 strain in OPA Assay.

FIG. 10: S. pn. 39 CP-CRM₁₉₇ and S. pn. 39 CP-TT conjugates sera canmediate killing of S. pn. 10A strain in OPA Assay.

FIG. 11: S. pn. 10A CP-CRM₁₉₇ conjugate sera can mediate killing of S.pn. 39 and 10A strains in OPA Assay.

FIG. 12: 20 v PnC vaccine sera can mediate killing of S. pn. 39 strainin OPA Assay. 20v PnC Contains S. pn. 10A CP-CRM197 Conjugate.

FIG. 13: 20 v PnC vaccine sera can mediate killing of S. pn. 10A Strainsin OPA Assay. 20v PnC Contains S. pn. 10A CP-CRM₁₉₇ Conjugate.

1 Immunogenic Compositions of the Invention

Immunogenic compositions of the present invention will typicallycomprise conjugated capsular saccharide antigens (also namedglycoconjugates), wherein the saccharides are derived from serotypes ofS. pneumoniae.

Preferably, the number of S. pneumoniae capsular saccharides can rangefrom 1 serotype (or “v”, valences) to 26 different serotypes (26v). Inone embodiment there is one serotype. In one embodiment there are 2different serotypes. In one embodiment there are 3 different serotypes.In one embodiment there are 4 different serotypes. In one embodimentthere are 5 different serotypes. In one embodiment there are 6 differentserotypes. In one embodiment there are 7 different serotypes. In oneembodiment there are 8 different serotypes. In one embodiment there are9 different serotypes. In one embodiment there are 10 differentserotypes. In one embodiment there are 11 different serotypes. In oneembodiment there are 12 different serotypes. In one embodiment there are13 different serotypes. In one embodiment there are 14 differentserotypes. In one embodiment there are 15 different serotypes. In oneembodiment there are 16 different serotypes. In an embodiment there are17 different serotypes. In an embodiment there are 18 differentserotypes. In an embodiment there are 19 different serotypes. In anembodiment there are 20 different serotypes. In an embodiment there are21 different serotypes. In an embodiment there are 22 differentserotypes. In an embodiment there are 23 different serotypes. In anembodiment there are 24 different serotypes. In an embodiment there are25 different serotypes. In an embodiment there are 26 differentserotypes. The capsular saccharides are conjugated to a carrier proteinto form glycoconjugates as described here below.

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharides could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 or more differentsaccharides conjugated to it) [see for instance WO 2004/083251].

In a preferred embodiment though, the saccharides are each individuallyconjugated to different molecules of the protein carrier (each moleculeof protein carrier only having one type of saccharide conjugated to it).In said embodiment, the capsular saccharides are said to be individuallyconjugated to the carrier protein.

For the purposes of the invention the term ‘glycoconjugate’ indicates acapsular saccharide linked covalently to a carrier protein. In oneembodiment a capsular saccharide is linked directly to a carrierprotein. In a second embodiment a bacterial saccharide is linked to aprotein through a spacer/linker.

1.1 Carrier Protein of the Invention

A component of the glycoconjugate of the invention is a carrier proteinto which the saccharide is conjugated. The terms “protein carrier” or“carrier protein” or “carrier” may be used interchangeably herein.Carrier proteins should be amenable to standard conjugation procedures.

In a preferred embodiment, the carrier protein of the glycoconjugates isselected in the group consisting of: DT (Diphtheria toxin), TT (tetanustoxid) or fragment C of TT, CRM₁₉₇ (a nontoxic but antigenicallyidentical variant of diphtheria toxin), the A chain of diphtheria toxinmutant CRM₁₉₇ (CN103495161), other DT mutants (such as CRM176, CRM228,CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102,CRM103 or CRM107, and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992);deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740;mutation of at least one or more residues Lys 516, Lys 526, Phe 530and/or Lys 534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017and 6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711,pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect Immun63:2706-2713) including ply detoxified in some fashion, for exampledPLY-GMBS (WO 2004/081515 and WO 2006/032499) or dPLY-formol, PhtX,including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtEare disclosed in WO 00/37105 and WO 00/39299) and fusions of Phtproteins for example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334,WO 03/054007, WO 2009/000826), OMPC (meningococcal outer membraneprotein—usually extracted from Neisseria meningitidis serogroup B(EP0372501), PorB (from N. meningitidis), PD (Haemophilus influenzaeprotein ID, see, e.g., EP0594610 B), or immunologically functionalequivalents thereof, synthetic peptides (EP0378881, EP0427347), heatshock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones(WO 91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (Falugi et al. (2001)Eur J Immunol 31:3816-3824) such as N19 protein (Baraldoi et al. (2004)Infect Immun 72:4884-4887) pneumococcal surface protein PspA (WO02/091998), iron uptake proteins (WO 01/72337), toxin A or B ofClostridium difficile (WO 00/61761), transferrin binding proteins,pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosaexotoxin A (in particular non-toxic mutants thereof (such as exotoxin Abearing a substution at glutamic acid 553 (Douglas et al. (1987) J.Bacteriol. 169(11):4967-4971)). Other proteins, such as ovalbumin,keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purifiedprotein derivative of tuberculin (PPD) also can be used as carrierproteins. Other suitable carrier proteins include inactivated bacterialtoxins such as cholera toxoid (e.g., as described in WO 2004/083251),Escherichia coli LT, E. coli ST, and exotoxin A from P. aeruginosa.

In a preferred embodiment, the carrier protein of the glycoconjugates isindependently selected from the group consisting of TT, DT, DT mutants(such as CRM₁₉₇), H. influenzae protein D, PhtX, PhtD, PhtDE fusions(particularly those described in WO 01/98334 and WO 03/054007),detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B ofC. difficile and PsaA.

In an embodiment, the carrier protein of the glycoconjugates of theinvention is DT (Diphtheria toxoid). In another embodiment, the carrierprotein of the glycoconjugates of the invention is TT (tetanus toxid).

In another embodiment, the carrier protein of the glycoconjugates of theinvention is PD (H. influenzae protein D, see, e.g., EP0594610 B).

In another embodiment, the carrier protein of the glycoconjugates of theinvention is CRM₁₉₇.

The CRM₁₉₇ protein is a nontoxic form of diphtheria toxin but isimmunologically indistinguishable from the diphtheria toxin. CRM₁₉₇ isproduced by Corynebacterium diphtheriae infected by the nontoxigenicphage β197^(tox−) created by nitrosoguanidine mutagenesis of thetoxigenic corynephage beta (Uchida et al. (1971) Nature New Biology233:8-11). The CRM₁₉₇ protein has the same molecular weight as thediphtheria toxin but differs therefrom by a single base change (guanineto adenine) in the structural gene. This single base change causes anamino acid substitution (glutamic acid for glycine) in the matureprotein and eliminates the toxic properties of diphtheria toxin. TheCRM₁₉₇ protein is a safe and effective T-cell dependent carrier forsaccharides. Further details about CRM₁₉₇ and production thereof can befound, e.g., in U.S. Pat. No. 5,614,382. In an embodiment, the capsularsaccharides of the invention are conjugated to CRM₁₉₇ protein or the Achain of CRM₁₉₇ (see CN103495161). In an embodiment, the capsularsaccharides of the invention are conjugated the A chain of CRM₁₉₇obtained via expression by genetically recombinant E. coli (seeCN103495161). In an embodiment, the capsular saccharides of theinvention are all conjugated to CRM₁₉₇. In an embodiment, the capsularsaccharides of the invention are all conjugated to the A chain ofCRM₁₉₇.

Accordingly, in frequent embodiments, the glycoconjugates of theinvention comprise CRM₁₉₇ as the carrier protein, wherein the capsularpolysaccharide is covalently linked to CRM₁₉₇.

1.2 Capsular Saccharide of the Invention

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. In frequentembodiments, the saccharide is a polysaccharide, in particular a S.pneumoniae capsular polysaccharide.

Capsular polysaccharides are prepared by standard techniques known tothose of ordinary skill in the art.

In the present invention, capsular polysaccharides may be prepared,e.g., from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39 of S.pneumoniae. Typically capsular polysaccharides are produced by growingeach S. pneumoniae serotype in a medium (e.g., in a soy-based medium),the polysaccharides are then prepared from the bacteria culture.Bacterial strains of S. pneumoniae used to make the respectivepolysaccharides that are used in the glycoconjugates of the inventionmay be obtained from established culture collections or clinicalspecimens.

The population of the organism (each S. pneumoniae serotype) is oftenscaled up from a seed vial to seed bottles and passaged through one ormore seed fermentors of increasing volume until production scalefermentation volumes are reached. At the end of the growth cycle thecells are lysed and the lysate broth is then harvested for downstream(purification) processing (see for example WO 2006/110381, WO2008/118752, and U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381,2008/0102498 and 2008/0286838).

The individual polysaccharides are typically purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography (see for example WO 2006/110352 and WO 2008/118752).

Purified polysaccharides may be activated (e.g., chemically activated)to make them capable of reacting (e.g., either directly to the carrierprotein of via a linker such as an eTEC spacer) and then incorporatedinto glycoconjugates of the invention, as further described herein.

S. pneumoniae capsular polysaccharides comprise repeatingoligosaccharide units which may contain up to 8 sugar residues.

In an embodiment, capsular saccharide of the invention may be oneoligosaccharide unit, or a shorter than native length saccharide chainof repeating oligosaccharide units.

In an embodiment, capsular saccharide of the invention is one repeatingoligosaccharide unit of the relevant serotype.

In an embodiment, capsular saccharide of the invention may beoligosaccharides. Oligosaccharides have a low number of repeat units(typically 5-15 repeat units) and are typically derived synthetically orby hydrolysis of polysaccharides.

Preferably though, all of the capsular saccharides of the presentinvention and in the immunogenic compositions of the present inventionare polysaccharides. High molecular weight capsular polysaccharides areable to induce certain antibody immune responses due to the epitopespresent on the antigenic surface. The isolation and purification of highmolecular weight capsular polysaccharides is preferably contemplated foruse in the conjugates, compositions and methods of the presentinvention.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 5 kDa and 4,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 10 kDaand 4,000 kDa; between 50 kDa and 4,000 kDa; between 50 kDa and 3,000kDa; between 50 kDa and 2,000 kDa; between 50 kDa and 1,500 kDa; between50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500kDa; between 100 kDa and 4,000 kDa; between 100 kDa and 3,000 kDa; 100kDa and 2,000 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa;between 100 and 400 kDa; between 200 kDa and 4,000 kDa; between 200 kDaand 3,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,500kDa; between 200 kDa and 1,000 kDa; or between 200 kDa and 500 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of between 70 kDa to 150 kDa; 80 kDa to 160 kDa; 90 kDa to 250kDa; 100 kDa to 1,000; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDato 160 kDa; 150 kDa to 600 kDa; 200 kDa to 1,000 kDa; 200 kDa to 600kDa; 200 kDa to 400 kDa; 300 kDa to 1,000 KDa; 300 kDa to 600 kDa; 300kDa to 500 kDa or 500 kDa to 600 kDa. Any whole number integer withinany of the above ranges is contemplated as an embodiment of thedisclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.Mechanical or chemical sizing maybe employed. Chemical hydrolysis maybeconducted using acetic acid. Mechanical sizing maybe conducted usingHigh Pressure Homogenization Shearing. The molecular weight rangesmentioned above refer to purified polysaccharides before conjugation(e.g., before activation).

In a preferred embodiment the purified polysaccharides, are capsularpolysaccharide from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A,11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F or 39 ofS. pneumoniae, wherein the capsular polysaccharide has a molecularweight falling within one of the molecular weight ranges as describedhere above.

As used herein, the term “molecular weight” of polysaccharide or ofcarrier protein-polysaccharide conjugate refers to molecular weightcalculated by size exclusion chromatography (SEC) combined withmultiangle laser light scattering detector (MALLS).

In some embodiments, the pneumococcal saccharides from serotypes 9V,18C, 11A, 15B, 22F and/or 33F of the invention are O-acetylated. In someembodiments, the pneumococcal saccharides from serotypes 9V, 11A, 15B,22F and/or 33F of the invention are O-acetylated.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (see for exampleLemercinier et al. (1996) Carbohydrate Research 296:83-96, Jones et al.(2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247, WO2005/033148 and WO 00/56357). Another commonly used method is describedin Hestrin (1949) J. Biol. Chem. 180:249-261. Preferably, the presenceof O-acetyl groups is determined by ion-H PLC analysis.

The purified polysaccharides described herein are chemically activatedto make the saccharides capable of reacting with the carrier protein.These pneumococcal conjugates are prepared by separate processes andformulated into a single dosage formulation as described below.

1.3 Glycoconjugates of the Invention

The purified saccharides are chemically activated to make thesaccharides (i.e., activated saccharides) capable of reacting with thecarrier protein, either directly or via a linker. Once activated, eachcapsular saccharide is separately conjugated to a carrier protein toform a glycoconjugate. In one embodiment, each capsular saccharide isconjugated to the same carrier protein. The chemical activation of thesaccharides and subsequent conjugation to the carrier protein can beachieved by the activation and conjugation methods disclosed herein.

1.3.1 Pneumococcal Polysaccharide from S. pneumoniae Serotypes 1, 2, 3,4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A,19F, 20, 22F, 23F, and 33F

Capsular polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23Fand/or 33F of S. pneumoniae are prepared as disclosed above.

In an embodiment, the polysaccharides are activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer (linker) group to an amino group on the carrier protein(preferably CRM₁₉₇). For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example usingN-[γ-maleimidobutyrloxy]succinimide ester (GMBS)) or a haloacetylatedcarrier protein (for example using iodoacetimide, N-succinimidylbromoacetate (SBA; SIB), N-succinimidyl(4-iodoacetyl)aminobenzoate(SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB),N-succinimidyl iodoacetate (SIA), or succinimidyl3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein (e.g., CRM₁₉₇) using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F areprepared using CDAP chemistry. In an embodiment of the presentinvention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 8, 9V, 14, 18C, 19F, and 23F are prepared using CDAP chemistry. Inan embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 14, 18C, 19F, and 23Fare prepared using CDAP chemistry. In an embodiment of the presentinvention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 8, 9V, 14, 18C, 19A, 19F, and 23F are prepared using CDAP chemistry.In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 14, 18C, 19A, 19F, and23F are prepared using CDAP chemistry. In an embodiment of the presentinvention, the glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5,6A, 6B, 7F, 8, 9V, 14, 18C, 19A, 19F, and 23F are prepared using CDAPchemistry.

Other suitable techniques for conjugation use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S—NHS, EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (see Bethell et al. (1979) 1. Biol. Chern.254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followedby reaction with a protein to form a carbamate linkage. This may involvereduction of the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group, reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on a protein(CDI chemistry).

In an preferred embodiment, at least one of capsular polysaccharidesfrom serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F of S. pneumoniae isconjugated to the carrier protein by reductive amination (such asdescribed in U.S. Patent Appl. Pub. Nos. 2006/0228380, 2007/184072,2007/0231340 and 2007/0184071, WO 2006/110381, WO 2008/079653, and WO2008/143709).

In an embodiment of the present invention, the glycoconjugate from S.pneumoniae serotype 6A is prepared by reductive amination. In anembodiment of the present invention, the glycoconjugate from S.pneumoniae serotype 19A is prepared by reductive amination. In anembodiment of the present invention, the glycoconjugate from S.pneumoniae serotype 3 is prepared by reductive amination. In anembodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 6A and 19A are prepared by reductive amination. Inan embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 3, 6A and 19A are prepared by reductive amination.

In a preferred embodiment of the present invention, the glycoconjugatesfrom S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F areprepared by reductive amination. In an embodiment of the presentinvention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 6B,9V, 14, 18C, 19F and 23F are prepared by reductive amination. In anembodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F are preparedby reductive amination. In an embodiment of the present invention, theglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F and 23F are prepared by reductive amination. In an embodimentof the present invention, the glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F are prepared byreductive amination. In an embodiment of the present invention, theglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F are prepared by reductive amination. In anembodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F areall prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F and23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F and 23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F areall prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23Fand 33F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniaeserotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are all prepared byreductive amination.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide, (2) reduction of the activated polysaccharide and acarrier protein to form a conjugate. Before oxidation, thepolysaccharide is optionally hydrolyzed. Mechanical or chemicalhydrolysis maybe employed. Chemical hydrolysis maybe conducted usingacetic acid.

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and includes the various salts of periodate(e.g., sodium periodate and potassium periodate). In an embodiment thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxydized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In an embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in WO 2014/097099). In said reaction, the actualoxidant is the N-oxoammonium salt, in a catalytic cycle. In an aspect,said stable nitroxyl or nitroxide radical compound are piperidine-N-oxyor pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl ornitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-ChloroSuccinimide, N-Bromosuccinimide, N-lodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to an “activated polysaccharide”here below. The activated polysaccharide and the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized). In one embodiment theactivated polysaccharide and the carrier protein are co-lyophilized. Inanother embodiment the activated polysaccharide and the carrier proteinare lyophilized independently.

In one embodiment the lyophilization takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(so-called reductive amination), using a reducing agent. Reducing agentswhich are suitable include the cyanoborohydrides (such as sodiumcyanoborohydride, sodium triacetoxyborohydride or sodium or zincborohydride in the presence of Bronsted or Lewis acids), amine boranessuch as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol,dimethylamine-borane, t-BuMe^(i)PrN—BH₃, benzylamine-BH₃ or5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin. Inone embodiment the reducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent (e.g., selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES,MOPSO, BES, MOPS, DI PSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine orHEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5), inanother embodiment the reaction is carried out in aprotic solvent. In anembodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilized.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). Following the conjugation (the reduction reactionand optionally the capping), the glycoconjugates may be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration precipitation/elution, column chromatography(DEAE or hydrophobic interaction chromatography), and depth filtration.In an embodiment, the glycoconjugates are purified by diafilitration orion exchange chromatography or size exclusion chromatography.

In one embodiment the glycoconjugates are sterile filtered.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, and 23F areprepared using CDAP chemistry and the glycoconjugate from S. pneumoniaeserotype 6A is prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, and 23F areprepared using CDAP chemistry and the glycoconjugate from S. pneumoniaeserotype 19A is prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, and 23F areprepared using CDAP chemistry and the glycoconjugates from S. pneumoniaeserotype 6A and 19A are prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, and 23F areprepared using CDAP chemistry and the glycoconjugates from S. pneumoniaeserotype 3, 6A and 19A are prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23Fare prepared using CDAP chemistry and the glycoconjugate from S.pneumoniae serotype 6A is prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F, and 23Fare prepared using CDAP chemistry and the glycoconjugate from S.pneumoniae serotype 19A is prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F, and 23Fare prepared using CDAP chemistry and the glycoconjugates from S.pneumoniae serotype 6A and 19A are prepared by reductive amination.

In an embodiment of the present invention, the glycoconjugates from S.pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23Fare prepared using CDAP chemistry and the glycoconjugates from S.pneumoniae serotype 3, 6A and 19A are prepared by reductive amination.

In an embodiment, the glycoconjugates of the invention are preparedusing the eTEC conjugation, such as described in WO 2014/027302. Saidglycoconjugates comprise a saccharide covalently conjugated to a carrierprotein through one or more eTEC spacers, wherein the saccharide iscovalently conjugated to the eTEC spacer through a carbamate linkage,and wherein the carrier protein is covalently conjugated to the eTECspacer through an amide linkage. The eTEC linked glycoconjugates of theinvention may be represented by the general formula (I):

where the atoms that comprise the eTEC spacer are contained in thecentral box.

The eTEC spacer includes seven linear atoms (i.e.,—C(O)NH(CH₂)₂SCH₂C(O)—) and provides stable thioether and amide bondsbetween the saccharide and carrier protein. In said glycoconjugates ofthe invention, the saccharide may be a polysaccharide or anoligosaccharide. The carrier protein may be selected from any suitablecarrier as described herein or known to those of skill in the art. Infrequent embodiments, the saccharide is a polysaccharide. In some suchembodiments, the carrier protein is CRM₁₉₇.

In some embodiments, the glycoconjugate from S. pneumoniae serotypes 1,7F, 9V, and/or 18C of the invention are O-acetylated. In someembodiments, the glycoconjugate from S. pneumoniae serotypes 1, 7F and9V is O-acetylated and the glycoconjugate from S. pneumoniae serotype18C is de-O-acetylated.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 1comprise a saccharide which has a degree of O-acetylation of between 10and 100%, between 20 and 100%, between 30 and 100%, between 40 and 100%,between 50 and 100%, between 60 and 100%, between 70 and 100%, between75 and 100%, 80 and 100%, 90 and 100%, 50 and 90%, 60 and 90%, 70 and90% or 80 and 90%. In other embodiments, the degree of O-acetylation is≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 7Fcomprise a saccharide which has a degree of O-acetylation of between 10and 100%, between 20 and 100%, between 30 and 100%, between 40 and 100%,between 50 and 100%, between 60 and 100%, between 70 and 100%, between75 and 100%, 80 and 100%, 90 and 100%, 50 and 90%, 60 and 90%, 70 and90% or 80 and 90%. In other embodiments, the degree of O-acetylation is≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 9Vcomprise a saccharide which has a degree of O-acetylation of between 10and 100%, between 20 and 100%, between 30 and 100%, between 40 and 100%,between 50 and 100%, between 60 and 100%, between 70 and 100%, between75 and 100%, 80 and 100%, 90 and 100%, 50 and 90%, 60 and 90%, 70 and90% or 80 and 90%. In other embodiments, the degree of O-acetylation is≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 18Ccomprise a saccharide which has a degree of O-acetylation of between 10and 100%, between 20 and 100%, between 30 and 100%, between 40 and 100%,between 50 and 100%, between 60 and 100%, between 70 and 100%, between75 and 100%, 80 and 100%, 90 and 100%, 50 and 90%, 60 and 90%, 70 and90% or 80 and 90%. In other embodiments, the degree of O-acetylation is≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.Preferably though, the glycoconjugate from S. pneumoniae serotype 18C isde-O-acetylated. In some said embodiments, the glycoconjugate from S.pneumoniae serotype 18C comprise a saccharide which has a degree ofO-acetylation of between 0 and 50%, between 0 and 40%, between 0 and30%, between 0 and 20%, between 0 and 10%, between 0 and 5%, or between0 and 2%. In other embodiments, the degree of O-acetylation is ≤50%,≤40%, ≤30%, ≤20%, ≤10%, ≤5%, ≤2%, or ≤1%.

By % of O-acetylation it is meant the percentage of a given sacchariderelative to 100% (where each repeat unit is fully acetylated relative toits acetylated structure).

In some embodiments, the glycoconjugates of the present inventioncomprise a saccharide having a molecular weight of between 10 kDa and2,000 kDa. In other such embodiments, the saccharide has a molecularweight of between 50 kDa and 1,000 kDa. In other such embodiments, thesaccharide has a molecular weight of between 70 kDa and 900 kDa. Inother such embodiments, the saccharide has a molecular weight of between100 kDa and 800 kDa. In other such embodiments, the saccharide has amolecular weight of between 200 kDa and 600 kDa. In further suchembodiments, the saccharide has a molecular weight of 100 kDa to 1000kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDato 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa;150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa; 150 kDa to700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa; 200 kDa to 800kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to 500 kDa; 200 kDato 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa; 250 kDa to 900 kDa;250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to 600 kDa; 250 kDa to500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 1,000 kDa;300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700 kDa; 300 kDa to600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1,000 kDa;400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700 kDa; 400 kDa to600 kDa; 500 kDa to 600 kDa. Any whole number integer within any of theabove ranges is contemplated as an embodiment of the disclosure. In somesuch embodiments, the glycoconjugate is prepared using reductiveamination.

In some embodiments, the glycoconjugate of the invention has a molecularweight of between 400 kDa and 15,000 kDa; between 500 kDa and 10,000kDa; between 2,000 kDa and 10,000 kDa; between 3,000 kDa and 8,000 kDakDa; or between 3,000 kDa and 5,000 kDa. In other embodiments, theglycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa.In other embodiments, glycoconjugate has a molecular weight of between1,000 kDa and 8,000 kDa. In still other embodiments, the glycoconjugatehas a molecular weight of between 2,000 kDa and 8,000 kDa or between3,000 kDa and 7,000 kDa. In further embodiments, the glycoconjugate ofthe invention has a molecular weight of between 200 kDa and 20,000 kDa;between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDaand 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDa and 20,000kDa; between 500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa;between 500 kDa and 10,000 kDa; between 500 kDa and 7,500 kDa; between500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa; between 500 kDaand 4,000 kDa; between 500 kDa and 3,000 kDa; between 500 kDa and 2,000kDa; between 500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa;between 750 kDa and 20,000 kDa; between 750 kDa and 15,000 kDa; between750 kDa and 12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDaand 7,500 kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000kDa; between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa;between 750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 12,500 kDa; between1,000 kDa and 10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000kDa and 6,000 kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDaand 4,000 kDa; between 1,000 kDa and 2,500 kDa; between 2,000 kDa and15,000 kDa; between 2,000 kDa and 12,500 kDa; between 2,000 kDa and10,000 kDa; between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000kDa; between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa;or between 2,000 kDa and 3,000 kDa.

In further embodiments, the glycoconjugate of the invention has amolecular weight of between 3,000 kDa and 20,000 kDa; between 3,000 kDaand 15,000 kDa; between 3,000 kDa and 10,000 kDa; between 3,000 kDa and7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000 kDa and 20,000kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDa and 12,500 kDa;between 4,000 kDa and 10,000 kDa; between 4,000 kDa and 7,500 kDa;between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and 5,000 kDa.

In further embodiments, the glycoconjugate of the invention has amolecular weight of between 5,000 kDa and 20,000 kDa; between 5,000 kDaand 15,000 kDa; between 5,000 kDa and 10,000 kDa; between 5,000 kDa and7,500 kDa; between 6,000 kDa and 20,000 kDa; between 6,000 kDa and15,000 kDa; between 6,000 kDa and 12,500 kDa; between 6,000 kDa and10,000 kDa or between 6,000 kDa and 7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

In a preferred embodiment, the serotype 22F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 orabout 0.8 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the glycoconjugate comprises at least 0.6 mM acetate per mM serotype 22Fpolysaccharide. In a preferred embodiment, the glycoconjugate comprisesat least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the serotype 33F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 33F capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 33Fcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 15B capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 15Bcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMglycerol per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the serotype 15B glycoconjugate of the invention comprisesat least 0.5, 0.6 or 0.7 mM glycerol per mM serotype 15B capsularpolysaccharide. In a preferred embodiment, the serotype 15Bglycoconjugate of the invention comprises at least 0.6 mM glycerol permM serotype 15B capsular polysaccharide. In a preferred embodiment, theserotype 15B glycoconjugate of the invention comprises at least 0.7 mMglycerol per mM serotype 15B capsular polysaccharide.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.3, 0.5, 0.6, 1.0, 1.4, 1.8, 2.2, 2.6,3.0, 3.4, 3.8, 4.2, 4.6 or about 5.0 mM acetate per mM serotype 11Apolysaccharide. In a preferred embodiment, the serotype 11Aglycoconjugate comprises at least 1.8, 2.2 or 2.6 mM acetate per mMserotype 11A polysaccharide. In an embodiment, the glycoconjugatecomprises at least 0.6 mM acetate per mM serotype 11A polysaccharide. Ina preferred embodiment, the serotype 11A glycoconjugate of the inventioncomprises at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0, 3.4, 3.8, 4.2 orabout 4.6 mM acetate per mM serotype 11A polysaccharide and less thanabout 5.0 mM acetate per mM serotype 11A polysaccharide. In anembodiment, the serotype 11A glycoconjugate of the invention comprisesat least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, or about 3.0 mM acetate per mMserotype 11A polysaccharide and less than about 3.4 mM acetate per mMserotype 11A polysaccharide. In an embodiment, the serotype 11Aglycoconjugate of the invention comprises at least 0.6, 1.0, 1.4, 1.8,2.2, 2.6, or about 3.0 mM acetate per mM serotype 11A polysaccharide andless than about 3.3 mM acetate per mM serotype 11A polysaccharide. Anyof the above number is contemplated as an embodiment of the disclosure.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or about 1.0 mM glycerol per mM serotype 11A polysaccharide. In apreferred embodiment, the serotype 11A glycoconjugate comprises at least0.2, 0.3 or 0.4 mM glycerol per mM serotype 11A polysaccharide. In apreferred embodiment, the serotype 11A glycoconjugate of the inventioncomprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or about 0.9mM glycerol per mM serotype 11A polysaccharide and less than about 1.0mM glycerol per mM serotype 11A polysaccharide. In a preferredembodiment, the serotype 11A glycoconjugate of the invention comprisesat least 0.3, 0.4, 0.5, 0.6, or about 0.7 mM glycerol per mM serotype11A polysaccharide and less than about 0.8 mM glycerol per mM serotype11A polysaccharide. Any of the above number is contemplated as anembodiment of the disclosure.

Another way to characterize the glycoconjugates of the invention is bythe number of lysine residues in the carrier protein (e.g., CRM₁₉₇) thatbecome conjugated to the saccharide which can be characterized as arange of conjugated lysines (degree of conjugation). The evidence forlysine modification of the carrier protein, due to covalent linkages tothe polysaccharides, can be obtained by amino acid analysis usingroutine methods known to those of skill in the art. Conjugation resultsin a reduction in the number of lysine residues recovered, compared tothe carrier protein starting material used to generate the conjugatematerials. In a preferred embodiment, the degree of conjugation of theglycoconjugate of the invention is between 2 and 15, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the glycoconjugate of the invention is about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14 or about 15. In a preferred embodiment,the degree of conjugation of the glycoconjugate of the invention isbetween 4 and 7. In some such embodiments, the carrier protein isCRM₁₉₇.

The glycoconjugates of the invention may also be characterized by theratio (weight/weight) of saccharide to carrier protein. In someembodiments, the ratio of polysaccharide to carrier protein in theglycoconjugate (w/w) is between 0.5 and 3 (e.g., about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In otherembodiments, the saccharide to carrier protein ratio (w/w) is between0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments,the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Ina preferred embodiment, the ratio of capsular polysaccharide to carrierprotein in the conjugate is between 0.9 and 1.1. In some suchembodiments, the carrier protein is CRM₁₉₇.

The glycoconjugates and immunogenic compositions of the invention maycontain free saccharide that is not covalently conjugated to the carrierprotein, but is nevertheless present in the glycoconjugate composition.The free saccharide may be non-covalently associated with (i.e.,non-covalently bound to, adsorbed to, or entrapped in or with) theglycoconjugate.

In a preferred embodiment, the glycoconjugate comprises less than about50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide comparedto the total amount of polysaccharide. In a preferred embodiment theglycoconjugate comprises less than about 25% of free polysaccharidecompared to the total amount of polysaccharide. In a preferredembodiment the glycoconjugate comprises less than about 20% of freepolysaccharide compared to the total amount of polysaccharide. In apreferred embodiment the glycoconjugate comprises less than about 15% offree polysaccharide compared to the total amount of polysaccharide.

The glycoconjugates may also be characterized by their molecular sizedistribution (K_(d)). Size exclusion chromatography media (CL-4B) can beused to determine the relative molecular size distribution of theconjugate. Size Exclusion Chromatography (SEC) is used in gravity fedcolumns to profile the molecular size distribution of conjugates. Largemolecules excluded from the pores in the media elute more quickly thansmall molecules. Fraction collectors are used to collect the columneluate. The fractions are tested colorimetrically by saccharide assay.For the determination of K_(d), columns are calibrated to establish thefraction at which molecules are fully excluded (V₀), (K_(d)=0), and thefraction representing the maximum retention (V_(i)), (K_(d)=1). Thefraction at which a specified sample attribute is reached (V_(e)), isrelated to K_(d) by the expression, K_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 30% of the glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 40% of the glycoconjugate has a K_(d) below orequal to 0.3 in a CL-4B column. In a preferred embodiment, at least 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the glycoconjugate has a K_(d) below orequal to 0.3 in a CL-4B column. In a preferred embodiment, between 50%and 80% of the glycoconjugate has a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, between 65% and 80% of theglycoconjugate has a K_(d) below or equal to 0.3 in a CL-4B column. Thefrequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing theglycoconjugates of the invention. For example, in some embodiments, atleast one covalent linkage between the carrier protein and thepolysaccharide occurs for every 4 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 10saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 15 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every25 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage via an eTEC spacer between the CRM₁₉₇ and the polysaccharideoccurs at least once in every 4, 10, 15 or 25 saccharide repeat units ofthe polysaccharide.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; every 4 to 25saccharide repeat units or every 2 to 25 saccharide repeat units. Infrequent embodiments, the carrier protein is CRM₁₉₇.

In another embodiment, at least one linkage between carrier protein andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide. In an embodiment, the carrier protein is CRM₁₉₇. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

1.3.2 Pneumococcal Polysaccharide from S. pneumoniae Serotype 39

S. pneumoniae 39 capsular polysaccharides comprise repeatingoligosaccharide units which contain 7 sugar residues and are composedof: D-Galp, D-GalpNAc, D-Galf and Ribitol in the molar ratios of 3:1:2:1(Petersen et al., Carbohydr. Res. 2014, 395, 38-46; Bush et al., J.Bacteriol. 2014, 196, 3271-3278). S. pneumoniae 39 capsularpolysaccharides are O-acetylated at two positions of terminal β-Galf.The molecular weight of a repeating unit of Pn 39 polysaccharide (sodiumsalt) is 1334 g/mol or 1334 Da when O-acetylated at said two positionsand 1250 g/mol or 1250 Da for de-O-acetylated polysaccharide.

In an embodiment, serotype 39 capsular saccharide of the invention maybe oligosaccharides. Oligosaccharides have a low number of repeat units(typically) and are typically derived synthetically or by hydrolysis ofpolysaccharides.

In such embodiment, serotype 39 saccharide of the invention may be asshort as one oligosaccharide unit (7 sugar residues, 1250 to 1334 Da).In another embodiment, serotype 39 capsular saccharide of the inventionis 2 to 15 repeat units long (2.5-20 kDa).

Serotype 39 saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 39 S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 39 before conjugation have a molecular weight of between 1.25kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 10 kDa and 2,000 kDa. In one embodiment, thecapsular polysaccharide has a molecular weight of between 50 kDa and1,000 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 70 kDa and 900 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 100 kDaand 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600 kDa; 250kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 600kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa; 500 kDato 600 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

S. pneumoniae 39 capsular polysaccharides is O-acetylated at twopositions of terminal β-Galf (Petersen et al., Carbohydr. Res. 2014,395, 38-46; Bush et al., J. Bacteriol. 2014, 196, 3271-3278) andtherefore may contain up to two O-acetyl groups per polysacchariderepeating unit.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (see for exampleLemercinier et al. (1996) Carbohydrate Research 296:83-96; Jones et al.(2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247; WO2005/033148 and WO 00/56357). Another commonly used method is describedin Hestrin, S. (1949) J. Biol. Chem. 180:249-261. Preferably, thepresence of O-acetyl groups is determined by ion-H PLC analysis.

The presence of O-acetyl in a purified, isolated or activated serotype39 capsular polysaccharide or in a serotype 39 polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofrepeat unit of said polysaccharide (counting the O-acetyl group only,i.e. excluding the N-acetyl group of the D-GalpNAc moiety) or as thenumber of O-acetyl group per polysaccharide repeating unit.

In an embodiment, the isolated serotype 39 capsular polysaccharidecomprises at least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 O-acetyl group perpolysaccharide repeating unit of said serotype 39 capsularpolysaccharide.

Preferably, the isolated serotype 39 capsular polysaccharide comprisesat least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0O-acetyl group per polysaccharide repeating unit of said serotype 39capsular polysaccharide. In a preferred embodiment, the isolatedserotype 39 capsular polysaccharide comprises about 0.8 mM, about 0.9 mMor about 1 mM O-acetyl group per polysaccharide repeating unit of saidserotype 39 capsular polysaccharide.

In another embodiment, the isolated serotype 39 capsular polysaccharideis de-O-acetylated. De-O-acetylation can be achieved prior toconjugation for example by treatment of the polysaccharide with mildbase (e.g. 0.1MNH₄OH).

In such said embodiment, the isolated serotype 39 capsularpolysaccharide comprises less than 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or lessthan 2 O-acetyl group per polysaccharide repeating unit of said serotype39 capsular polysaccharide. Preferably in said embodiment, the isolatedserotype 39 capsular polysaccharide comprises less than 0.01, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or less than 1.0 O-acetyl group perpolysaccharide repeating unit of said serotype 39 capsularpolysaccharide. Even more preferably, in such said embodiment, theisolated serotype 39 capsular polysaccharide comprises less than 0.01,0.1, 0.2, 0.3, 0.4 or less than 0.5 O-acetyl group per polysacchariderepeating unit of said serotype 39 capsular polysaccharide. In anembodiment, the serotype 39 glycoconjugates are obtained by activatingpolysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester (CDAP chemistry). The activatedpolysaccharide may be coupled directly (direct CDAP chemistry) or via aspacer (linker) group (indirect CDAP chemistry) to an amino group on thecarrier protein. For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide, SIB,SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein using carbodiimide (e.g., EDAC or EDC)chemistry via a carboxyl group on the protein carrier. Such conjugatesare described for example in WO 93/15760, WO 95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein (CDI chemistry).

In preferred embodiments, the serotype 39 glycoconjugates of theinvention are prepared using reductive amination.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 39comprises a saccharide which has a degree of O-acetylation of between 10and 100%, between 20 and 100%, between 30 and 100%, between 40 and 100%,between 50 and 100%, between 60 and 100%, between 70 and 100%, between75 and 100%, 80 and 100%, 90 and 100%, 95 and 100%, 50 and 90%, 60 and90%, 70 and 90%, 80 and 90%, 50 and 95%, 60 and 95%, 70 and 95%, 80 and95%, 85 and 95%, 90 and 95%, 50 and 98%, 60 and 98%, 70 and 98%, 80 and98%, 85 and 98%, 90 and 99% or 95 and 98%. In other embodiments, thedegree of O-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%,≥80%, ≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotype 39is de-O-acetylated. In some said embodiments, the glycoconjugate from S.pneumoniae serotype 39 comprise a saccharide which has a degree ofO-acetylation of between 0 and 50%, between 0 and 40%, between 0 and30%, between 0 and 20%, between 0 and 10%, between 0 and 5%, or between0 and 2%. In other embodiments, the degree of O-acetylation is ≤50%,≤40%, ≤30%, ≤20%, ≤10%, ≤5%, ≤2%, or ≤1%.

By % of O-acetylation it is meant the percentage of a given sacchariderelative to 100% (where D-GalpNAc moiety of each repeat unit is fullyacetylated at two positions).

In some embodiments, the serotype 39 glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between1.25 kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 10 kDa and 2,000 kDa. In other suchembodiments, the saccharide has a molecular weight of between 50 kDa and2,000 kDa. In further such embodiments, the saccharide has a molecularweight of between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa;between 50 kDa and 1,300 kDa; between 750 kDa and 1,250 kDa; between 50kDa and 750 kDa; between 100 kDa and 1000 kDa; between 500 kDa and 2,000kDa; between 750 kDa and 1,750 kDa; between 1000 kDa and 1,500 kDa;between 1000 kDa and 1,250 kDa; between 200 kDa and 2,000 kDa; between200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDaand 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750kDa; or between 200 kDa and 500 kDa; between 10 kDa and 500 kDa; between50 kDa and 250 kDa; between 50 kDa and 150 kDa or between 50 kDa and 125kDa. In some such embodiments, the serotype 39 glycoconjugates areprepared using reductive amination. In some such embodiments, theserotype 39 glycoconjugates are prepared using direct or indirect CDAPchemistry.

In some such embodiments, the serotype 39 glycoconjugates are preparedusing CDI chemistry.

In some embodiments, the serotype 39 glycoconjugate of the invention hasa molecular weight of between 50 kDa and 30,000 kDa. In otherembodiments, the serotype 39 glycoconjugate has a molecular weight ofbetween 50 kDa and 25,000 kDa. In other embodiments, the serotype 39glycoconjugate has a molecular weight of between 500 kDa and 20,000 kDa.In other embodiments, the serotype 39 glycoconjugate has a molecularweight of between 50 kDa and 15,000 kDa. In other embodiments, theserotype 39 glycoconjugate has a molecular weight of between 500 kDa and30,000 kDa, 500 kDa and 25,000 kDa, 500 kDa and 15,000 kDa, between 500kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; or between 3,000kDa and 8,000 kDa. In other embodiments, the serotype 39 glycoconjugatehas a molecular weight of between 1,000 kDa and 10,000 kDa. In otherembodiments, the serotype 39 glycoconjugate has a molecular weight ofbetween 1000 kDa and 8,000 kDa. In still other embodiments, the theserotype 39 glycoconjugate has a molecular weight of between 2,000 kDaand 8,000 kDa or between 3,000 kDa and 7,000 kDa. In furtherembodiments, the serotype 39 glycoconjugate of the invention has amolecular weight of between 200 kDa and 30,000 kDa; between 200 kDa and25,000 kDa; between 200 kDa and 20,000 kDa; between 200 kDa and 15,000kDa; between 200 kDa and 10,000 kDa; between 200 kDa and 7,500 kDa;between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa; between200 kDa and 1,000 kDa; between 500 kDa and 30,000 kDa; between 500 kDaand 25,000 kDa; between 500 kDa and 20,000 kDa; between 500 kDa and15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDa and 10,000kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000 kDa;between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa; between500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between 500 kDaand 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDa and 30,000kDa; between 750 kDa and 25,000 kDa; between 750 kDa and 20,000 kDa;between 750 kDa and 15,000 kDa; between 750 kDa and 12,500 kDa; between750 kDa and 10,000 kDa; between 750 kDa and 7,500 kDa; between 750 kDaand 6,000 kDa; between 750 kDa and 5,000 kDa; between 750 kDa and 4,000kDa; between 750 kDa and 3,000 kDa; between 750 kDa and 2,000 kDa;between 750 kDa and 1,500 kDa; between 1,000 kDa and 30,000 kDa; between1,000 kDa and 25,000 kDa; between 1,000 kDa and 20,000 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 12,500 kDa; between1,000 kDa and 10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000kDa and 6,000 kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDaand 4,000 kDa; between 1,000 kDa and 2,500 kDa; between 2,000 kDa and30,000 kDa; between 2,000 kDa and 25,000 kDa; between 2,000 kDa and20,000 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000kDa.

In further embodiments, the serotype 39 glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 30,000 kDa; between3,000 kDa and 25,000 kDa; between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 30,000 kDa; between 4,000 kDa and 25,000 kDa; between 4,000 kDaand 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDa and12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 39 glycoconjugate of theinvention has a molecular weight of between 5,000 kDa and 30,000 kDa;between 5,000 kDa and 25,000 kDa; between 5,000 kDa and 20,000 kDa;between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa orbetween 5,000 kDa and 7,500 kDa. In further embodiments, the serotype 39glycoconjugate of the invention has a molecular weight of between 6,000kDa and 30,000 kDa; between 6,000 kDa and 25,000 kDa; between 6,000 kDaand 20,000 kDa; between 6,000 kDa and 15,000 kDa; between 6,000 kDa and10,000 kDa or between 6,000 kDa and 7,500 kDa. In further embodiments,the serotype 39 glycoconjugate of the invention has a molecular weightof between 7,000 kDa and 30,000 kDa; between 7,000 kDa and 25,000 kDa;between 7,000 kDa and 20,000 kDa; between 7,000 kDa and 15,000 kDa;between 7,000 kDa and 10,000 kDa or between 7,000 kDa and 8,000 kDa. Infurther embodiments, the serotype 39 glycoconjugate of the invention hasa molecular weight of between 8,000 kDa and 30,000 kDa; between 8,000kDa and 25,000 kDa; between 8,000 kDa and 20,000 kDa; between 8,000 kDaand 15,000 kDa; or between 8,000 kDa and 10,000 kDa.

Any whole number integer within any of the above ranges is contemplatedas an embodiment of the disclosure. The molecular weight of theglycoconjugate is measured by SEC-MALLS.

In an embodiment, the serotype 39A glycoconjugate of the inventioncomprises at least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 O-acetyl group perpolysaccharide repeating unit of said serotype 39 polysaccharide. In apreferred embodiment, the serotype 39A glycoconjugate of the inventioncomprises at least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or1.0 O-acetyl group per polysaccharide repeating unit of said serotype 39polysaccharide. In a preferred embodiment, the glycoconjugate comprisesat least 0.8, 0.9 or 0.95 O-acetyl group per polysaccharide repeatingunit of serotype 39 polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.8 O-acetyl group per polysacchariderepeating unit of serotype 39 polysaccharide. In a preferred embodiment,the glycoconjugate comprises at least 0.9 O-acetyl group perpolysaccharide repeating unit of serotype 39 polysaccharide.

In another embodiment, the isolated serotype 39 capsular polysaccharideis de-Oacetylated. De-Oacetylation can be achieved prior to conjugationfor example by treatment of the polysaccharide with mild base (e.g.0.1MNH₄OH).

In some embodiments, the glycoconjugate from S. pneumoniae serotype 39is de-O-acetylated. In some said embodiments, the glycoconjugate from S.pneumoniae serotype 39 comprise less than 0.01, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 orless than 2 O-acetyl group per polysaccharide repeating unit of saidserotype 39 capsular polysaccharide. Preferably, in some saidembodiments, the glycoconjugate from S. pneumoniae serotype 39 compriseless than 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or less than1.0 O-acetyl group per polysaccharide repeating unit of said serotype 39capsular polysaccharide. Even more preferably, in such said embodiment,the glycoconjugate comprises less than 0.01, 0.1, 0.2, 0.3, 0.4 or lessthan 0.5 O-acetyl group per polysaccharide repeating unit of serotype 39capsular polysaccharide.

Another way to characterize the serotype 39 glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 39glycoconjugate is between 2 and 19, between 2 and 17, 2 and 15, between2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2and 5, between 2 and 4, between 3 and 19, between 3 and 17, between 3and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3and 6, between 3 and 5, between 3 and 4, between 5 and 19, between 5 and17, between 5 and 15, between 5 an 10, between 8 and 19, between 8 and17, between 8 and 15, between 8 and 12, between 10 and 19, between 10and 17, between 10 and 15,between 10 and 12, between 12 and 19, between12 and 17 or between 12 and 15. In a preferred embodiment, the degree ofconjugation of the serotype 39 glycoconjugate is between 3 and 6. In apreferred embodiment, the carrier protein is CRM₁₉₇. In anotherpreferred embodiment, the carrier protein is TT.

The serotype 39 glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7,about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0,about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about2.7, about 2.8, about 2.9 or about 3.0). In a preferred embodiment, theratio of serotype 39 saccharide to carrier protein in the conjugate isbetween 0.5 and 2.0, 0.5 and 1.5, 0.5 and 1.0, 1.0 and 1.5 or 1.0 and2.0.

In a preferred embodiment, the ratio of serotype 39 polysaccharide tocarrier protein in the conjugate is between 0.4 and 0.9. In a preferredembodiment, the ratio of serotype 39 capsular polysaccharide to carrierprotein in the conjugate is between 0.4 and 0.8 (e.g., about 0.4, about0.5 about 0.6, about 0.7, or about 0.8). In some such embodiments, thecarrier protein is CRM₁₉₇.

The serotype 39 glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 39 glycoconjugates of the inventioncomprise less than about 50% free saccharide, less than about 45% freesaccharide, less than about 40% free saccharide, less than about 35%free saccharide, less than about 30% free saccharide, less than about25% free saccharide, less than about 20% free saccharide, less thanabout 15% free saccharide, less than about 10% free saccharide, or lessthan about 5% free saccharide relative to the total amount of 39saccharide. Preferably, serotype 39 the glycoconjugate comprises lessthan 15% free saccharide, more preferably less than 10% free saccharide,and still more preferably, less than 5% of free saccharide.

The serotype 39 glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above.

In a preferred embodiment, at least 30% of the serotype 39glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 40% of the serotype 39glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, or 85% of the serotype 39 glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 60% of the serotype 39 glycoconjugateshave a K_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 50% and 80% of the serotype 39 glycoconjugates ofthe invention have a K_(d) below or equal to 0.3 in a CL-4B column.

1.4Combination of Glycoconjugates of the Invention

In an embodiment the immunogenic composition of the invention comprisesany of the glycoconjugates disclosed herein.

1.4.1 Combinations of Glycoconjugates

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate from S. pneumoniae serotype 39.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the two following S. pneumoniaeserotypes: 39 and 4, 39 and 6B, 39 and 14, 39 and 18C, 39 and 19F or 39and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the eigth following S. pneumoniaeserotypes: 39, 4, 6B, 9V, 14, 18C, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the nine following S. pneumoniaeserotypes: 39, 9V, 1, 4, 6B, 14, 18C, 19F and 23F; 39, 9V, 4, 5, 6B, 14,18C, 19F, and 23F; 39, 9V, 4, 6B 7F, 14, 18C, 19F, and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the eleven following S.pneumoniae serotypes: 39, 1, 5, 4, 6B, 7F, 9V, 14, 18C, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the twelve following S.pneumoniae serotypes: 39, 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F;39, 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the thirteen following S.pneumoniae serotypes: 39, 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the fourteen following S.pneumoniae serotypes: 39, 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate from S. pneumoniae serotype 10A.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the two following S. pneumoniaeserotypes: 10A and 4, 10A and 6B, 10A and 14, 10A and 18C, 10A and 19For 10A and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the eighth following S.pneumoniae serotypes: 10A, 4, 6B, 9V, 14, 18C, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the nine following S. pneumoniaeserotypes: 10A, 9V, 1, 4, 6B, 14, 18C, 19F and 23F; 10A, 9V, 4, 5, 6B,14, 18C, 19F, and 23F; 10A, 9V, 4, 6B 7F, 14, 18C, 19F, and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the eleven following S.pneumoniae serotypes: 10A, 1, 5, 4, 6B, 7F, 9V, 14, 18C, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the twelve following S.pneumoniae serotypes: 10A, 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and23F; 10A, 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the thirteen following S.pneumoniae serotypes: 10A, 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19Fand 23F.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the thirteen following S.pneumoniae serotypes: 10A, 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19Fand 23F.

1.4.2 Additional Combinations of Glycoconjugates

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 15B.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 22F.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 33F.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises (if not present) in addition at least one glycoconjugateof S. pneumoniae serotype 10A.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 11A.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of S. pneumoniaeserotype 12F.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of thetwo following S. pneumoniae serotypes:

15B and 22F,

15B and 33F,

15B and 12F,

15B and 10A,

15B and 11A,

15B and 8,

22F and 33F,

22F and 12F,

22F and 10A,

22F and 11A,

22F and 8,

33F and 12F,

33F and 10A,

33F and 11A,

33F and 8,

12F and 10A,

12F and 11A,

12F and 8,

10A and 11A,

10A and 8, or

11A and 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of thethree following S. pneumoniae serotypes:

15B and 22F and 33F,

15B and 22F and 12F,

15B and 22F and 10A,

15B and 22F and 11A,

15B and 22F and 8,

15B and 33F and 12F,

15B and 33F and 10A,

15B and 33F and 11A,

15B and 33F and 8,

15B and 12F and 10A,

15B and 12F and 11A,

15B and 12F and 8,

15B and 10A and 11A,

15B and 10A and 8,

15B and 11A and 8,

22F and 33F and 12F,

22F and 33F and 10A,

22F and 33F and 11A,

22F and 33F and 8,

22F and 12F and 10A,

22F and 12F and 11A,

22F and 12F and 8,

22F and 10A and 11A,

22F and 10A and 8,

22F and 11A and 8,

33F and 12F and 10A,

33F and 12F and 11A,

33F and 12F and 8,

33F and 10A and 11A,

33F and 10A and 8,

33F and 11A and 8,

12F and 10A and 11A,

12F and 10A and 8,

12F and 11A and 8, or

10A and 11A and 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of thefour following S. pneumoniae serotypes:

15B and 22F and 33F and 12F,

15B and 22F and 33F and 10A,

15B and 22F and 33F and 11A,

15B and 22F and 33F and 8,

15B and 22F and 12F and 10A,

15B and 22F and 12F and 11A,

15B and 22F and 12F and 8,

15B and 22F and 10A and 11A,

15B and 22F and 10A and 8,

15B and 22F and 11A and 8,

15B and 33F and 12F and 10A,

15B and 33F and 12F and 11A,

15B and 33F and 12F and 8,

15B and 33F and 10A and 11A,

15B and 33F and 10A and 8,

15B and 33F and 11A and 8,

15B and 12F and 10A and 11A,

15B and 12F and 10A and 8,

15B and 12F and 11A and 8,

15B and 10A and 11A and 8,

22F and 33F and 12F and 10A,

22F and 33F and 12F and 11A,

22F and 33F and 12F and 8,

22F and 33F and 10A and 11A,

22F and 33F and 10A and 8,

22F and 33F and 11A and 8,

22F and 12F and 10A and 11A,

22F and 12F and 10A and 8,

22F and 12F and 11A and 8,

22F and 10A and 11A and 8,

33F and 12F and 10A and 11A,

33F and 12F and 10A and 8,

33F and 12F and 11A and 8,

33F and 10A and 11A and 8 or

12F and 10A and 11A and 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of thefive following S. pneumoniae serotypes:

15B and 22F and 33F and 12F and 10A,

15B and 22F and 33F and 12F and 11A,

15B and 22F and 33F and 12F and 8,

15B and 22F and 33F and 10A and 11A,

15B and 22F and 33F and 10A and 8,

15B and 22F and 33F and 11A and 8,

15B and 22F and 12F and 10A and 11A,

15B and 22F and 12F and 10A and 8,

15B and 22F and 12F and 11A and 8,

15B and 22F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A,

15B and 33F and 12F and 10A and 8,

15B and 33F and 12F and 11A and 8,

15B and 33F and 10A and 11A and 8,

15B and 12F and 10A and 11A and 8,

22F and 33F and 12F and 10A and 11A,

22F and 33F and 12F and 10A and 8,

22F and 33F and 12F and 11A and 8,

22F and 33F and 10A and 11A and 8,

22F and 12F and 10A and 11A and 8 or

33F and 12F and 10A and 11A and 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of thesix following S. pneumoniae serotypes:

15B and 22F and 33F and 12F and 10A and 11A,

15B and 22F and 33F and 12F and 10A and 8,

15B and 22F and 33F and 12F and 11A and 8,

15B and 22F and 33F and 10A and 11A and 8,

15B and 22F and 12F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A and 8 or

22F and 33F and 12F and 10A and 11A and 8.

In an embodiment any of the immunogenic composition defined at 1.4.1above comprises in addition at least one glycoconjugate of each of theseven following S. pneumoniae serotypes: 15B and 22F and 33F and 12F and10A and 11A and 8.

In an embodiment any of the immunogenic composition above comprises inaddition glycoconjugates from S. pneumoniae serotype 2.

In an embodiment any of the immunogenic composition above comprises inaddition glycoconjugates from S. pneumoniae serotype 17F.

In an embodiment any of the immunogenic composition above comprises inaddition glycoconjugates from S. pneumoniae serotype 20.

In an embodiment any of the immunogenic composition above comprises inaddition glycoconjugates from S. pneumoniae serotype 15C.

In an embodiment any of the immunogenic composition above comprises inaddition glycoconjugates from S. pneumoniae serotype 9N.

Preferably, all the glycoconjugates of the above immunogenic compositionare individually conjugated to the carrier protein.

In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 39 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 22F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 33F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 15B is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 12F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 10A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 11A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 8 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and23F are conjugated to CRM₁₉₇. In an embodiment of any of the aboveimmunogenic composition, the glycoconjugates from S. pneumoniaeserotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In an embodiment of anyof the above immunogenic composition, the glycoconjugates from S.pneumoniae serotypes 6A and 19A are conjugated to CRM₁₉₇. In anembodiment of any of the above immunogenic composition, theglycoconjugates from S. pneumoniae serotype 3 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 2 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 17F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 20 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 15C is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 9N is conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of the above immunogeniccompositions are all individually conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 39 ofany of the above immunogenic composition is individually conjugated toPD.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogeniccompositions are individually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of the above immunogenic compositions is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of the above immunogenic compositions is conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogeniccompositions are individually conjugated to PD, the glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT and the glycoconjugatefrom S. pneumoniae serotype 19F is conjugated to DT.

In an embodiment the above immunogenic composition comprises from 7 to26 different serotypes of S. pneumoniae. In one embodiment the aboveimmunogenic composition comprises glycoconjugates from 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 differentserotypes.

In an embodiment the above immunogenic composition comprises from 7 to20 different serotypes of S. pneumoniae. In one embodiment the aboveimmunogenic composition comprises glycoconjugates from 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 different serotypes. In oneembodiment the above immunogenic composition comprises glycoconjugatesfrom 16 or 20 different serotypes.

Preferably, all the glycoconjugates of the immunogenic composition ofthe invention are individually conjugated to the carrier protein. In anembodiment, the glycoconjugates of the immunogenic composition above areindividually conjugated to CRM₁₉₇.

Preferably, any of the immunogenic compositions defined at 1.4.1 or1.4.2 above that comprise at least one glycoconjugate of S. pneumoniaeserotype 10A do not comprise capsular saccharide from S. pneumoniaeserotype 39. Therefore, preferably, any of the immunogenic compositionsdefined at 1.4.1 or 1.4.2 above that comprise at least oneglycoconjugate of S. pneumoniae serotype 10A do not compriseglycoconjugate of S. pneumoniae serotype 39.

Preferably, any of the immunogenic compositions defined at 1.4.1 or1.4.2 above that comprise at least one glycoconjugate of S. pneumoniaeserotype 39 do not comprise capsular saccharide from S. pneumoniaeserotype 10A. Therefore, preferably, any of the immunogenic compositionsdefined at 1.4.1 or 1.4.2 above that comprise at least oneglycoconjugate of S. pneumoniae serotype 39 do not compriseglycoconjugate of S. pneumoniae serotype 10A.

After conjugation of the capsular polysaccharide to the carrier protein,the glycocopnjugates are purified (enriched with respect to the amountof polysaccharide-protein conjugate) by a variety of techniques. Thesetechniques include concentration/diafiltration operations,precipitation/elution, column chromatography, and depth filtration. See,e.g., U.S. Appl. Publication No. 2007/0184072 and WO 2008/079653. Afterthe individual glycoconjugates are purified, they are compounded toformulate the immunogenic composition of the present invention.

2 Dosage of the Immunogenic Compositions 2.1 Polysaccharide Amount

The amount of glycoconjugate(s) in each dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccines. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented.

The amount of a particular glycoconjugate in an immunogenic compositioncan be calculated based on total polysaccharide for that conjugate(conjugated and non-conjugated). For example, a glycoconjugate with 20%free polysaccharide will have about 80 μg of conjugated polysaccharideand about 20 μg of non-conjugated polysaccharide in a 100 μgpolysaccharide dose. The amount of glycoconjugate can vary dependingupon the streptococcal serotype. The saccharide concentration can bedetermined by the uronic acid assay.

The “immunogenic amount” of the different polysaccharide components inthe immunogenic composition, may diverge and each may comprise about 1.0μg, about 2.0 μg, about 3.0 μg, about 4.0 μg, about 5.0 μg, about 6.0μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, about 10.0 μg, about 15.0μg, about 20.0 μg, about 30.0 μg, about 40.0 μg, about 50.0 μg, about60.0 μg, about 70.0 μg, about 80.0 μg, about 90.0 μg, or about 100.0 μgof any particular polysaccharide antigen.

Generally, each dose will comprise 0.1 μg to 100 μg of polysaccharidefor a given serotype, particularly 0.5 μg to 20 μg, more particularly 1μg to 10 μg, and even more particularly 2 μg to 5 μg. Any whole numberinteger within any of the above ranges is contemplated as an embodimentof the disclosure.

In an embodiment, each dose will comprise 1 μg, 2 μg, 3 μg, 4 μg, 5 μg,6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg or 20 μg of polysaccharide for agiven serotype.

2.2 Carrier Amount

Generally, each dose will comprise 5 μg to 150 μg of carrier protein,particularly 10 μg to 100 μg of carrier protein, more particularly 15 μgto 100 μg of carrier protein, more particularly 25 to 75 μg of carrierprotein, more particularly 30 μg to 70 μg of carrier protein, moreparticularly 30 to 60 μg of carrier protein, more particularly 30 μg to50 μg of carrier protein and even more particularly 40 to 60 μg ofcarrier protein. In an embodiment, said carrier protein is CRM₁₉₇.

In an embodiment, each dose will comprise about 25 μg, about 26 μg,about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg,about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg,about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg,about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about65 μg, about 66 μg, about 67 μg, 68 μg, about 69 μg, about 70 μg, about71 μg, about 72 μg, about 73 μg, about 74 μg or about 75 μg of carrierprotein. In an embodiment, said carrier protein is CRM₁₉₇.

3 Further Antigens

Immunogenic compositions of the invention comprise conjugated S.pneumoniae saccharide antigens (glycoconjugates). They may also furtherinclude antigens from other pathogens, particularly from bacteria and/orviruses. Preferred further antigens are selected from: a diphtheriatoxoid (D), a tetanus toxoid (T), a pertussis antigen (P), which istypically acellular (Pa), a hepatitis B virus (HBV) surface antigen(HBsAg), a hepatitis A virus (HAV) antigen, a conjugated Haemophilusinfluenzae type b capsular saccharide (Hib), inactivated poliovirusvaccine (IPV).

In an embodiment, the immunogenic compositions of the invention compriseD-T-Pa. In an embodiment, the immunogenic compositions of the inventioncomprise D-T-Pa-Hib, D-T-Pa-IPV or D-T-Pa-HBsAg. In an embodiment, theimmunogenic compositions of the invention comprise D-T-Pa-HBsAg-IPV orD-T-Pa-HBsAg-Hib. In an embodiment, the immunogenic compositions of theinvention comprise D-T-Pa-HBsAg-IPV-Hib.

Pertussis antigens: Bordetella pertussis causes whooping cough.Pertussis antigens in vaccines are either cellular (whole cell, in theform of inactivated B. pertussis cells) or acellular. Preparation ofcellular pertussis antigens is well documented (e.g., it may be obtainedby heat inactivation of phase I culture of B. pertussis). Preferably,however, the invention uses acellular antigens. Where acellular antigensare used, it is preferred to use one, two or (preferably) three of thefollowing antigens: (1) detoxified pertussis toxin (pertussis toxoid, orPT); (2) filamentous hemagglutinin (FHA); (3) pertactin (also known asthe 69 kiloDalton outer membrane protein). FHA and pertactin may betreated with formaldehyde prior to use according to the invention. PT ispreferably detoxified by treatment with formaldehyde and/orglutaraldehyde. Acellular pertussis antigens are preferably adsorbedonto one or more aluminum salt adjuvants. As an alternative, they may beadded in an unadsorbed state. Where pertactin is added then it ispreferably already adsorbed onto an aluminum hydroxide adjuvant. PT andFHA may be adsorbed onto an aluminum hydroxide adjuvant or an aluminumphosphate. Adsorption of all of PT, FHA and pertactin to aluminumhydroxide is most preferred.

Inactivated poliovirus vaccine: Poliovirus causes poliomyelitis. Ratherthan use oral poliovirus vaccine, preferred embodiments of the inventionuse IPV. Prior to administration to patients, polioviruses must beinactivated, and this can be achieved by treatment with formaldehyde.Poliomyelitis can be caused by one of three types of poliovirus. Thethree types are similar and cause identical symptoms, but they areantigenically different and infection by one type does not protectagainst infection by others. It is therefore preferred to use threepoliovirus antigens in the invention: poliovirus Type 1 (e.g., Mahoneystrain), poliovirus Type 2 (e.g., MEF-1 strain), and poliovirus Type 3(e.g., Saukett strain). The viruses are preferably grown, purified andinactivated individually, and are then combined to give a bulk trivalentmixture for use with the invention.

Diphtheria toxoid: Corynebacterium diphtheriae causes diphtheria.Diphtheria toxin can be treated (e.g., using formalin or formaldehyde)to remove toxicity while retaining the ability to induce specificanti-toxin antibodies after injection. These diphtheria toxoids are usedin diphtheria vaccines. Preferred diphtheria toxoids are those preparedby formaldehyde treatment. The diphtheria toxoid can be obtained bygrowing C. diphtheriae in growth medium, followed by formaldehydetreatment, ultrafiltration and precipitation. The toxoided material maythen be treated by a process comprising sterile filtration and/ordialysis. The diphtheria toxoid is preferably adsorbed onto an aluminumhydroxide adjuvant.

Tetanus toxoid: Clostridium tetani causes tetanus. Tetanus toxin can betreated to give a protective toxoid. The toxoids are used in tetanusvaccines. Preferred tetanus toxoids are those prepared by formaldehydetreatment. The tetanus toxoid can be obtained by growing C. tetani ingrowth medium, followed by formaldehyde treatment, ultrafiltration andprecipitation. The material may then be treated by a process comprisingsterile filtration and/or dialysis.

Hepatitis A virus antigens: Hepatitis A virus (HAV) is one of the knownagents which causes viral hepatitis. A preferred HAV component is basedon inactivated virus, and inactivation can be achieved by formalintreatment.

Hepatitis B virus (HBV) is one of the known agents which causes viralhepatitis. The major component of the capsid is a protein known as HBVsurface antigen or, more commonly, HBsAg, which is typically a 226-aminoacid polypeptide with a molecular weight of ˜24 kDa. All existinghepatitis B vaccines contain HBsAg, and when this antigen isadministered to a normal vaccine it stimulates the production ofanti-HBsAg antibodies which protect against HBV infection.

For vaccine manufacture, HBsAg has been made in two ways: purificationof the antigen in particulate form from the plasma of chronic hepatitisB carriers or expression of the protein by recombinant DNA methods(e.g., recombinant expression in yeast cells). Unlike native HBsAg(i.e., as in the plasma-purified product), yeast-expressed HBsAg isgenerally non-glycosylated, and this is the most preferred form of HBsAgfor use with the invention.

Conjugated Haemophilus influenzae type b antigens: Haemophilusinfluenzae type b (Hib) causes bacterial meningitis. Hib vaccines aretypically based on the capsular saccharide antigen, the preparation ofwhich is well documented. The Hib saccharide can be conjugated to acarrier protein in order to enhance its immunogenicity, especially inchildren. Typical carrier proteins are tetanus toxoid, diphtheriatoxoid, CRM197, H. influenzae protein D, and an outer membrane proteincomplex from serogroup B meningococcus. The saccharide moiety of theconjugate may comprise full-length polyribosylribitol phosphate (PRP) asprepared from Hib bacteria, and/or fragments of full-length PRP. Hibconjugates may or may not be adsorbed to an aluminum salt adjuvant.

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup A capsular saccharide(MenA), a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

4 Adjuvant(s)

In some embodiments, the immunogenic compositions disclosed herein mayfurther comprise at least one adjuvant (e.g., one, two or threeadjuvants). The term “adjuvant” refers to a compound or mixture thatenhances the immune response to an antigen. Antigens may act primarilyas a delivery system, primarily as an immune modulator or have strongfeatures of both. Suitable adjuvants include those suitable for use inmammals, including humans.

Examples of known suitable delivery-system type adjuvants that can beused in humans include, but are not limited to, alum (e.g., aluminumphosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate,liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5%w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)),water-in-oil emulsions such as Montanide, andpoly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

In an embodiment, the immunogenic compositions disclosed herein comprisealuminum salts (alum) as adjuvant (e.g., aluminum phosphate, aluminumsulfate or aluminum hydroxide). In a preferred embodiment, theimmunogenic compositions disclosed herein comprise aluminum phosphate oraluminum hydroxide as adjuvant. In an embodiment, the immunogeniccompositions disclosed herein comprise from 0.1 mg/mL to 1 mg/mL or from0.2 mg/mL to 0.3 mg/ml of elemental aluminum in the form of aluminumphosphate. In an embodiment, the immunogenic compositions disclosedherein comprise about 0.25 mg/mL of elemental aluminum in the form ofaluminum phosphate. Examples of known suitable immune modulatory typeadjuvants that can be used in humans include, but are not limited to,saponin extracts from the bark of the Aquilla tree (QS21, Quil A), TLR4agonists such as MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylatedMPL) or GLA-AQ, LT/CT mutants, cytokines such as the variousinterleukins (e.g., IL-2, IL-12) or GM-CSF, and the like.

Examples of known suitable immune modulatory type adjuvants with bothdelivery and immune modulatory features that can be used in humansinclude, but are not limited to ISCOMS (see, e.g., Spönder et al. (1998)J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EMwhich is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications including but not limited to animalexperimentation, one can use Complete Freund's Adjuvant (CFA), Freund'sIncomplete Adjuvant (IFA), Emulsigen,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and R1131, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further exemplary adjuvants to enhance effectiveness of the pneumococcalvaccines as disclosed herein include, but are not limited to: (1)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion, and (b) RIBI™ adjuvant system(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2%Tween 80, and one or more bacterial cell wall components such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants, suchas QS21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), Abisco®(Isconova, Sweden), or Iscomatrix® (Commonwealth Serum Laboratories,Australia), may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes), which ISCOMS may be devoid of additionaldetergent (e.g., WO 00/07621); (3) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins(e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636)),interferons (e.g., gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB-2220221,EP0689454), optionally in the substantial absence of alum when used withpneumococcal saccharides (see, e.g., WO 00/56358); (6) combinations of3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g.,EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g., WO99/52549); (8) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO 01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (9) a saponin and an immunostimulatory oligonucleotide (e.g.,a CpG oligonucleotide) (WO 00/62800); (10) an immunostimulant and aparticle of metal salt (see e.g., WO00/23105); (11) a saponin and anoil-in-water emulsion e.g., WO 99/11241; (12) a saponin (e.g.,QS21)+3dMPL+IM2 (optionally+a sterol) e.g., WO 98/57659; (13) othersubstances that act as immunostimulating agents to enhance the efficacyof the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpGoligonucleotide as used herein refers to an immunostimulatory CpGoligodeoxynucleotide (CpG ODN), and accordingly these terms are usedinterchangeably unless otherwise indicated. Immunostimulatory CpGoligodeoxynucleotides contain one or more immunostimulatory CpG motifsthat are unmethylated cytosine-guanine dinucleotides, optionally withincertain preferred base contexts. The methylation status of the CpGimmunostimulatory motif generally refers to the cytosine residue in thedinucleotide. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is an oligonucleotide which contains a5′ unmethylated cytosine linked by a phosphate bond to a 3′ guanine, andwhich activates the immune system through binding to Toll-like receptor9 (TLR-9). In another embodiment the immunostimulatory oligonucleotidemay contain one or more methylated CpG dinucleotides, which willactivate the immune system through TLR9 but not as strongly as if theCpG motif(s) was/were unmethylated. CpG immunostimulatoryoligonucleotides may comprise one or more palindromes that in turn mayencompass the CpG dinucleotide. CpG oligonucleotides have been describedin a number of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise any of the CpG Oligonucleotide described atpages 3, lines 22, to page 12, line 36, of WO 2010/125480.

Different classes of CpG immunostimulatory oligonucleotides have beenidentified. These are referred to as A, B, C and P class, and aredescribed in greater detail at pages 3, lines 22, to page 12, line 36,of WO 2010/125480. Methods of the invention embrace the use of thesedifferent classes of CpG immunostimulatory oligonucleotides.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise an A class CpG oligonucleotide. In anembodiment of the present invention, the immunogenic compositions asdisclosed herein comprise a B class CpG Oligonucleotide.

The B class CpG oligonucleotide sequences of the invention are thosebroadly described above as well as disclosed in published WO 96/02555,WO 98/18810, and in U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068. Exemplary sequences include but arenot limited to those disclosed in these latter applications and patents.

In an embodiment, the “B class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID NO: 1) 5′ TCGTCGTTTTTCGGTGCTTTT 3′,  or (SEQ ID NO: 2) 5′TCGTCGTTTTTCGGTCGTTTT 3′,  or (SEQ ID NO: 3) 5′TCGTCGTTTTGTCGTTTTGTCGTT 3′,  or (SEQ ID NO: 4) 5′TCGTCGTTTCGTCGTTTTGTCGTT 3′,  or (SEQ ID NO: 5) 5′TCGTCGTTTTGTCGTTTTTTTCGA 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

Some non-limiting examples of B-Class oligonucleotides include:

(SEQ ID NO: 6) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′, or(SEQ ID NO: 7) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′, or(SEQ ID NO: 8) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T* T 3′,or (SEQ ID NO: 9) 5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3′, or (SEQ ID NO: 10) 5′T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G* A 3′.

wherein “*” refers to a phosphorothioate bond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a C class CpG oligonucleotide.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a P class CpG oligonucleotide.

In one embodiment the oligonucleotide includes at least onephosphorothioate linkage.

In another embodiment all internucleotide linkages of theoligonucleotide are phosphorothioate linkages. In another embodiment theoligonucleotide includes at least one phosphodiester-like linkage. Inanother embodiment the phosphodiester-like linkage is a phosphodiesterlinkage. In another embodiment a lipophilic group is conjugated to theoligonucleotide. In one embodiment the lipophilic group is cholesterol.

In an embodiment, all the internucleotide linkage of the CpGoligonucleotides disclosed herein are phosphodiester bonds (“soft”oligonucleotides, as described in WO 2007/026190). In anotherembodiment, CpG oligonucleotides of the invention are rendered resistantto degradation (e.g., are stabilized). A “stabilized oligonucleotide”refers to an oligonucleotide that is relatively resistant to in vivodegradation (e.g., via an exo- or endo-nuclease). Nucleic acidstabilization can be accomplished via backbone modifications.Oligonucleotides having phosphorothioate linkages provide maximalactivity and protect the oligonucleotide from degradation byintracellular exo- and endo-nucleases.

The immunostimulatory oligonucleotides may have a chimeric backbone,which have combinations of phosphodiester and phosphorothioate linkages.For purposes of the instant invention, a chimeric backbone refers to apartially stabilized backbone, wherein at least one internucleotidelinkage is phosphodiester or phosphodiester-like, and wherein at leastone other internucleotide linkage is a stabilized internucleotidelinkage, wherein the at least one phosphodiester or phosphodiester-likelinkage and the at least one stabilized linkage are different. When thephosphodiester linkage is preferentially located within the CpG motifsuch molecules are called “semi-soft” as described in WO 2007/026190.

Other modified oligonucleotides include combinations of phosphodiester,phosphorothioate, methylphosphonate, methylphosphorothioate,phosphorodithioate, and/or p-ethoxy linkages.

Mixed backbone modified ODN may be synthesized as described in WO2007/026190. The size of the CpG oligonucleotide (i.e., the number ofnucleotide residues along the length of the oligonucleotide) also maycontribute to the stimulatory activity of the oligonucleotide. Forfacilitating uptake into cells, CpG oligonucleotide of the inventionpreferably have a minimum length of 6 nucleotide residues.Oligonucleotides of any size greater than 6 nucleotides (even many kblong) are capable of inducing an immune response if sufficientimmunostimulatory motifs are present, because larger oligonucleotidesare degraded inside cells. In certain embodiments, the CpGoligonucleotides are 6 to 100 nucleotides long, preferentially 8 to 30nucleotides long. In important embodiments, nucleic acids andoligonucleotides of the invention are not plasmids or expressionvectors.

In an embodiment, the CpG oligonucleotide disclosed herein comprisesubstitutions or modifications, such as in the bases and/or sugars asdescribed at paragraphs 134 to 147 of WO 2007/026190.

In an embodiment, the CpG oligonucleotide of the present invention ischemically modified. Examples of chemical modifications are known to theskilled person and are described, for example in Uhlmann et al. (1990)Chem. Rev. 90:543; S. Agrawal, Ed., Humana Press, Totowa, USA 1993;Crooke. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; andHunziker et al., (1995) Mod. Synth. Methods 7:331-417. Anoligonucleotide according to the invention may have one or moremodifications, wherein each modification is located at a particularphosphodiester internucleoside bridge and/or at a particular β-D-riboseunit and/or at a particular natural nucleoside base position incomparison to an oligonucleotide of the same sequence which is composedof natural DNA or RNA.

In some embodiments of the invention, CpG-containing nucleic acids mightbe simply mixed with immunogenic carriers according to methods known tothose skilled in the art (see, e.g., WO 03/024480).

In a particular embodiment of the present invention, any of theimmunogenic composition disclosed herein comprises from 2 μg to 100 mgof CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpGoligonucleotide, preferably from 0.2 mg to 10 mg CpG oligonucleotide,preferably from 0.3 mg to 5 mg CpG oligonucleotide, preferably from 0.3mg to 5 mg CpG oligonucleotide, even preferably from 0.5 mg to 2 mg CpGoligonucleotide, even preferably from 0.75 mg to 1.5 mg CpGoligonucleotide. In a preferred embodiment, any of the immunogeniccomposition disclosed herein comprises about 1 mg CpG oligonucleotide.

5 Formulation

The immunogenic compositions of the invention may be formulated inliquid form (i.e., solutions or suspensions) or in a lyophilized form.Liquid formulations may advantageously be administered directly fromtheir packaged form and are thus ideal for injection without the needfor reconstitution in aqueous medium as otherwise required forlyophilized compositions of the invention.

Formulation of the immunogenic composition of the present invention canbe accomplished using art-recognized methods. For instance, theindividual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

The present disclosure provides an immunogenic composition comprisingany of combination of glycoconjugates disclosed herein and apharmaceutically acceptable excipient, carrier, or diluent.

In an embodiment, the immunogenic composition of the invention is inliquid form, preferably in aqueous liquid form.

Immunogenic compositions of the disclosure may comprise one or more of abuffer, a salt, a divalent cation, a non-ionic detergent, acryoprotectant such as a sugar, and an anti-oxidant such as a freeradical scavenger or chelating agent, or any multiple combinationsthereof.

In an embodiment, the immunogenic composition of the invention comprisesa buffer. In an embodiment, said buffer has a pKa of about 3.5 to about7.5. In some embodiments, the buffer is phosphate, succinate, histidineor citrate. In certain embodiments, the buffer is succinate at a finalconcentration of 1 mM to 10 mM. In one particular embodiment, the finalconcentration of the succinate buffer is about 5 mM.

In an embodiment, the immunogenic composition of the invention comprisesa salt. In some embodiments, the salt is selected from the groupsconsisting of magnesium chloride, potassium chloride, sodium chlorideand a combination thereof. In one particular embodiment, the salt issodium chloride. In one particular embodiment, the immunogeniccomposition of the invention comprises sodium chloride at 150 mM.

In an embodiment, the immunogenic compositions of the invention comprisea surfactant. In an embodiment, the surfactant is selected from thegroup consisting of polysorbate 20 (TWEEN™20), polysorbate 40(TWEEN™40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65),polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-1 01,TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine,triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate(PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL),soy lecithin and a poloxamer. In one particular embodiment, thesurfactant is polysorbate 80. In some said embodiment, the finalconcentration of polysorbate 80 in the formulation is at least 0.0001%to 10% polysorbate 80 weight to weight (w/w). In some said embodiments,the final concentration of polysorbate 80 in the formulation is at least0.001% to 1% polysorbate 80 weight to weight (w/w). In some saidembodiments, the final concentration of polysorbate 80 in theformulation is at least 0.01% to 1% polysorbate 80 weight to weight(w/w). In other embodiments, the final concentration of polysorbate 80in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09% or 0.1% polysorbate 80 (w/w). In another embodiment, thefinal concentration of the polysorbate 80 in the formulation is 1%polysorbate 80 (w/w).

In certain embodiments, the immunogenic composition of the invention hasa pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even morepreferably a pH of 5.8 to 6.0.

In one embodiment, the present invention provides a container filledwith any of the immunogenic compositions disclosed herein. In oneembodiment, the container is selected from the group consisting of avial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, anampoule, a cartridge and a disposable pen. In certain embodiments, thecontainer is siliconized.

In an embodiment, the container of the present invention is made ofglass, metals (e.g., steel, stainless steel, aluminum, etc.) and/orpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In an embodiment, the container of the present invention is made ofglass.

In one embodiment, the present invention provides a syringe filled withany of the immunogenic compositions disclosed herein. In certainembodiments, the syringe is siliconized and/or is made of glass.

A typical dose of the immunogenic composition of the invention forinjection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1mL, even more preferably a volume of about 0.5 mL.

Therefore the container or syringe as defined above is filed with avolume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even morepreferably a volume of about 0.5 mL of any of the immunogeniccomposition defined herein.

6 Ability of the Immunogenic Compositions of the Invention to ElicitCross-Reactive Antibodies

In an embodiment, the immunogenic composition of the invention is ableto elicit IgG antibodies in human which are capable of binding S.pneumoniae serotypes 10A and/or 39 polysaccharide as determined by ELISAassay.

In the ELISA (Enzyme-linked Immunosorbent Assay) method, antibodies fromthe sera of vaccinated subjects are incubated with polysaccharides whichhave been adsorbed to a solid support. The bound antibodies are detectedusing enzyme-conjugated secondary detection antibodies.

In an embodiment said ELISA assay is the standardized ELISA assay asdefined by the WHO in the “Training Manual For Enzyme LinkedImmunosorbent Assay For The Quantitation Of Streptococcus PneumoniaeSerotype Specific IgG (Pn PS ELISA).” (available athttp://www.vaccine.uab.edu/ELISA %20protocol.pdf, accessed on Mar. 31,2014).

The ELISA measures type specific IgG anti-S. pneumoniae capsularpolysaccharide (PS) antibodies present in human serum. When dilutions ofhuman sera are added to type-specific capsular PS-coated microtiterplates, antibodies specific for that capsular PS bind to the microtiterplates. The antibodies bound to the plates are detected using a goatanti-human IgG alkaline phosphatase-labeled antibody followed by ap-nitrophenyl phosphate substrate. The optical density of the coloredend product is proportional to the amount of anticapsular PS antibodypresent in the serum.

In an embodiment, the immunogenic composition of the invention is ableto elicit IgG antibodies in human which are capable of binding S.pneumoniae serotypes 10A polysaccharide at a concentration of at least0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml as determinedby ELISA assay.

In an embodiment, the immunogenic composition of the invention is ableto elicit IgG antibodies in human which are capable of binding S.pneumoniae serotypes 39 polysaccharide at a concentration of at least0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml as determinedby ELISA assay.

In an embodiment, the immunogenic composition of the invention is ableto elicit functional antibodies in humans which are capable of killingS. pneumoniae serotype 10A and/or 39 as determined by in vitroopsonophagocytic assay (OPA) (see e.g. WO2015110941). In an embodiment,the immunogenic composition of the invention is able to elicitfunctional antibodies in humans which are capable of killing S.pneumoniae serotype 10A and 39 as determined by in vitroopsonophagocytic assay (OPA).

The pneumococcal opsonophagocytic assay (OPA), which measures killing ofS. pneumoniae cells by phagocytic effector cells in the presence offunctional antibody and complement, is considered to be an importantsurrogate for evaluating the effectiveness of pneumococcal vaccines.

In vitro opsonophagocytic assay (OPA) can be conducted by incubatingtogether a mixture of Streptococcus pneumoniae cells, a heat inactivatedhuman serum to be tested, differentiated HL-60 cells (phagocytes) and anexogenous complement source (e.g., baby rabbit complement).Opsonophagocytosis proceeds during incubation and bacterial cells thatare coated with antibody and complement are killed uponopsonophagocytosis. Colony forming units (cfu) of surviving bacteriathat escape from opsonophagocytosis are determined by plating the assaymixture. The OPA titer is defined as the reciprocal dilution thatresults in a 50% reduction in bacterial count over control wells withouttest serum. The OPA titer is interpolated from the two dilutions thatencompass this 50% killing cut-off.

An endpoint titer of 1:8 or greater is considered a positive result inthese killing type OPA.

In an embodiment, the immunogenic composition of the invention is ableto elicit a titer of at least 1:8 against S. pneumoniae serotype 10A inat least 50% of the subjects as determined by in vitro opsonophagocytickilling assay (OPA). In an embodiment, the immunogenic composition ofthe invention is able to elicit a titer of at least 1:8 against S.pneumoniae serotype 10A in at least 60%, 70%, 80%, or at least 90% ofthe subjects as determined by in vitro opsonophagocytic killing assay(OPA).

In an embodiment, the immunogenic composition of the invention is ableto elicit a titer of at least 1:8 against S. pneumoniae serotype 39 inat least 50% of the subjects as determined by in vitro opsonophagocytickilling assay (OPA). In an embodiment, the immunogenic composition ofthe invention is able to elicit a titer of at least 1:8 against S.pneumoniae serotype 39 in at least 60%, 70%, 80% or at least 90% of thesubjects as determined by in vitro opsonophagocytic killing assay (OPA).

In some embodiment, the subjects may have serotype specific OPA titersprior to pneumococcal vaccination due for example to natural exposuresto S. pneumoniae (e.g., in case of adult subjects).

Therefore, comparaison of OPA activity of pre- and post-immunizationserum with the immunogenic composition of the invention can be conductedand compared for their response to serotypes 10A and 39 to assess thepotential increase of responders.

In an embodiment the immunogenic composition of the inventionsignificantly increases the proportion of responders (i.e., individualwith a serum having a titer of at least 1:8 as determined by in vitroOPA) as compared to the pre-immunized population.

Therefore in an embodiment, the immunogenic composition of the inventionis able to significantly increase the proportion of responders againstS. pneumoniae serotype 10A (i.e., individual with a serum having a titerof at least 1:8 as determined by in vitro OPA) as compared to thepre-immunized population.

In an embodiment, the immunogenic composition of the invention is ableto significantly increase the proportion of responders against S.pneumoniae serotype 39 (i.e., individual with a serum having a titer ofat least 1:8 as determined by in vitro OPA) as compared to thepre-immunized population.

In an embodiment, the immunogenic composition of the invention is ableto significantly increase the proportion of responders against S.pneumoniae serotypes 10A and 39 (i.e., individual with a serum having atiter of at least 1:8 as determined by in vitro OPA) as compared to thepre-immunized population.

Comparison of OPA activity of pre- and post-immunization serum with theimmunogenic composition of the invention can also be done by comparingthe potential increase in OPA titers.

Therefore, comparison of OPA activity of pre- and post-immunizationserum with the immunogenic composition of the invention can be conductedand compared for their response to serotypes 10A and 39 to assess thepotential for increase in OPA titers.

In an embodiment the immunogenic compositions of the invention are ableto significantly increase the OPA titer of human subjects as compared tothe pre-immunized population.

Therefore in an embodiment, the immunogenic composition of the inventionis able to significantly increase the OPA titers of human subjectsagainst S. pneumoniae serotype 10A as compared to the pre-immunizedpopulation.

In an embodiment, the immunogenic composition of the invention is ableto significantly increase the OPA titers of human subjects against S.pneumoniae serotype 39 as compared to the pre-immunized population.

In an embodiment, the immunogenic composition of the invention is ableto significantly increase the OPA titers of human subjects against S.pneumoniae serotypes 10A and 39 as compared to the pre-immunizedpopulation.

7 Uses of the Immunogenic Compositions of the Invention

In an embodiment, the immunogenic compositions disclosed herein are foruse as a medicament.

The immunogenic compositions described herein may be used in varioustherapeutic or prophylactic methods for preventing, treating orameliorating a bacterial infection, disease or condition in a subject.In particular, immunogenic compositions described herein may be used toprevent, treat or ameliorate a S. pneumoniae infection, disease orcondition in a subject.

Thus in one aspect, the invention provides a method of preventing,treating or ameliorating an infection, disease or condition associatedwith S. pneumoniae in a subject, comprising administering to the subjectan immunologically effective amount of an immunogenic composition of theinvention.

In one aspect, the invention provides a method of preventing, treatingor ameliorating an infection, disease or condition associated with S.pneumoniae serotype 10A and/or 39 in a subject, comprising administeringto the subject an immunologically effective amount of an immunogeniccomposition of the invention.

In one aspect, the invention provides a method of inducing an immuneresponse to S. pneumoniae serotypes 10A and/or 39 in a subject,comprising administering to the subject an immunologically effectiveamount of an immunogenic composition of the invention.

In one aspect, the immunogenic compositions of the present invention arefor use in a method for preventing, treating or ameliorating aninfection, disease or condition caused by S. pneumoniae serotypes 10Aand/or 39 in a subject.

In an embodiment, any of the immunogenic composition disclosed herein isfor use in a method of immunizing a subject against infection by S.pneumoniae serotype 10A and/or 39.

In one aspect, the present invention is directed toward the use of theimmunogenic composition disclosed herein for the manufacture of amedicament for preventing, treating or ameliorating an infection,disease or condition caused by S. pneumoniae serotypes 10A and/or 39 ina subject.

In an embodiment, the present invention is directed toward the use ofthe immunogenic composition disclosed herein for the manufacture of amedicament for immunizing a subject against infection by S. pneumoniaeserotype 10A and/or 39.

In one aspect, the present invention provides a method for inducing animmune response to S. pneumoniae serotypes 10A and/or 39 in a subject.

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine. More particularly, the immunogenic compositionsdescribed herein may be used to prevent serotypes 10A and/or 39 S.pneumoniae infections in a subject. Thus in one aspect, the inventionprovides a method of preventing, an infection by serotypes 10A and/or 39S. pneumoniae in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition of theinvention. In some such embodiments, the infection is selected from thegroup consisting of pneumonia, sinusitis, otitis media, acute otitismedia, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis,osteomyelitis, septic arthritis, endocarditis, peritonitis,pericarditis, mastoiditis, cellulitis, soft tissue infection and brainabscess.

In one aspect, the subject to be vaccinated is a mammal, such as ahuman, cat, sheep, pig, horse, bovine or dog. Preferably, the subject tobe vaccinated is a human.

In one aspect, the immunogenic compositions disclosed herein are for usein a method of preventing, treating or ameliorating an infection,disease or condition associated S. pneumoniae with serotypes 10A and/or39 in a subject. In some such embodiments, the infection, disease orcondition is selected from the group consisting of pneumonia, sinusitis,otitis media, acute otitis media, meningitis, bacteremia, sepsis,pleural empyema, conjunctivitis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, softtissue infection and brain abscess.

In an aspect, the immunogenic composition disclosed herein are for usein a method of preventing, an infection by serotypes 10A and/or 39 of S.pneumoniae in a subject. In some such embodiments, the infection isselected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess. In one aspect, the subject to be vaccinatedis a mammal, such as a human, cat, sheep, pig, horse, bovine or dog.

In one aspect, the present invention is directed toward the use of theimmunogenic composition disclosed herein for the manufacture of amedicament for preventing, treating or ameliorating an infection,disease or condition associated S. pneumoniae with serotypes 10A and/or39 in a subject. In some such embodiments, the infection, disease orcondition is selected from the group consisting of pneumonia, sinusitis,otitis media, acute otitis media, meningitis, bacteremia, sepsis,pleural empyema, conjunctivitis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, softtissue infection and brain abscess.

In an aspect, the present invention is directed toward the use of theimmunogenic composition disclosed herein for the manufacture of amedicament for preventing, an infection by serotypes 10A and/or 39 of S.pneumoniae in a subject. In some such embodiments, the infection isselected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess. In one aspect, the subject to be vaccinatedis a mammal, such as a human, cat, sheep, pig, horse, bovine or dog.

The immunogenic compositions of the present invention can be used toprotect or treat a human susceptible to S. pneumoniae serotypes 10Aand/or 39 infection, by means of administering the immunogeniccompositions via a systemic or mucosal route.

In an embodiment, the immunogenic compositions disclosed herein areadministered by intramuscular, intraperitoneal, intradermal orsubcutaneous routes. In an embodiment, the immunogenic compositionsdisclosed herein are administered by intramuscular, intraperitoneal,intradermal or subcutaneous injection. In an embodiment, the immunogeniccompositions disclosed herein are administered by intramuscular orsubcutaneous injection.

8 Subject to be Treated with the Immunogenic Compositions of theInvention

As disclosed herein, the immunogenic compositions described herein maybe used in various therapeutic or prophylactic methods for preventing,treating or ameliorating a bacterial infection, disease or condition ina subject.

In a preferred embodiment, said subject is a human. In a most preferredembodiment, said subject is a newborn (i.e., under three months of age),an infant (i.e., from 3 months to one year of age) or a toddler (i.e.,from one year to four years of age).

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine.

In such embodiment, the subject to be vaccinated may be less than 1 yearof age. For example, the subject to be vaccinated can be about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11 or about 12 months of age. In an embodiment, the subject tobe vaccinated is about 2, 4 or 6 months of age. In another embodiment,the subject to be vaccinated is less than 2 years of age. For examplethe subject to be vaccinated can be about 12 to about 15 months of age.In some cases, as little as one dose of the immunogenic compositionaccording to the invention is needed, but under some circumstances, asecond, third or fourth dose may be given (see section 9 below).

In an embodiment of the present invention, the subject to be vaccinatedis a human 50 years of age or older, more preferably a human 55 years ofage or older. In an embodiment, the subject to be vaccinated is a human65 years of age or older, 70 years of age or older, 75 years of age orolder or 80 years of age or older.

In an embodiment the subject to be vaccinated is an immunocompromisedindividual, in particular a human. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal humoral or cellular defense to challenge byinfectious agents.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from a disease or condition that impairs theimmune system and results in an antibody response that is insufficientto protect against or treat pneumococcal disease.

In an embodiment, said disease is a primary immunodeficiency disorder.Preferably, said primary immunodeficiency disorder is selected from thegroup consisting of: combined T- and B-cell immunodeficiencies, antibodydeficiencies, well-defined syndromes, immune dysregulation diseases,phagocyte disorders, innate immunity deficiencies, autoinflammatorydisorders, and complement deficiencies. In an embodiment, said primaryimmunodeficiency disorder is selected from the one disclosed on page 24,line 11, to page 25, line 19, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from a diseaseselected from the groups consisting of: HIV-infection, acquiredimmunodeficiency syndrome (AIDS), cancer, chronic heart or lungdisorders, congestive heart failure, diabetes mellitus, chronic liverdisease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy,chronic bronchitis, emphysema, chronic obstructive pulmonary disease(COPD), spleen dysfunction (such as sickle cell disease), lack of spleenfunction (asplenia), blood malignancy, leukemia, multiple myeloma,Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome andasthma.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from malnutrition.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is taking a drug or treatmentthat lowers the body's resistance to infection. In an embodiment, saiddrug is selected from the one disclosed on page 26, line 33, to page 26,line 4, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is a smoker.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a white blood cell count(leukocyte count) below 5×10⁹ cells per liter, or below 4×10⁹ cells perliter, or below 3×10⁹ cells per liter, or below 2×10⁹ cells per liter,or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter, orbelow 0.3×10⁹ cells per liter, or below 0.1×10⁹ cells per liter.

White blood cell count (leukocyte count): The number of white bloodcells (WBC) in the blood. The WBC is usually measured as part of the CBC(complete blood count). White blood cells are the infection-fightingcells in the blood and are distinct from the red (oxygen-carrying) bloodcells known as erythrocytes. There are different types of white bloodcells, including neutrophils (polymorphonuclear leukocytes; PMN), bandcells (slightly immature neutrophils), T-type lymphocytes (T-cells),B-type lymphocytes (B-cells), monocytes, eosinophils, and basophils. Allthe types of white blood cells are reflected in the white blood cellcount. The normal range for the white blood cell count is usuallybetween 4,300 and 10,800 cells per cubic millimeter of blood. This canalso be referred to as the leukocyte count and can be expressed ininternational units as 4.3-10.8×10⁹ cells per liter.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from neutropenia. Ina particular embodiment of the present invention, the immunocompromisedsubject to be vaccinated has a neutrophil count below 2×10⁹ cells perliter, or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter,or below 0.1×10⁹ cells per liter, or below 0.05×10⁹ cells per liter.

A low white blood cell count or “neutropenia” is a conditioncharacterized by abnormally low levels of neutrophils in the circulatingblood. Neutrophils are a specific kind of white blood cell that helpprevent and fight infections. The most common reason that cancerpatients experience neutropenia is as a side effect of chemotherapy.Chemotherapy-induced neutropenia increases a patient's risk of infectionand disrupts cancer treatment.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a CD4+ cell count below500/mm3, or CD4+ cell count below 300/mm3, or CD4+ cell count below200/mm3, CD4+ cell count below 100/mm3, CD4+ cell count below 75/mm3, orCD4+ cell count below 50/mm3.

CD4 cell tests are normally reported as the number of cells in mm3.Normal CD4 counts are between 500 and 1600, and CD8 counts are between375 and 1100. CD4 counts drop dramatically in people with HIV.

In an embodiment of the invention, any of the immunocompromised subjectdisclosed herein is a human male or a human female.

9 Regimen

In some cases, as little as one dose of the immunogenic compositionaccording to the invention is needed, but under some circumstances, suchas conditions of greater immune deficiency, a second, third or fourthdose may be given. Following an initial vaccination, subjects canreceive one or several booster immunizations adequately spaced.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a single dose. In a particularembodiment, said single dose schedule is for healthy persons being atleast 2 years of age.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a multiple dose schedule. In aparticular embodiment, said multiple dose schedule consists of a seriesof 2 doses separated by an interval of about 1 month to about 2 months.In a particular embodiment, said multiple dose schedule consists of aseries of 2 doses separated by an interval of about 1 month, or a seriesof 2 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month to about 2 months.In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month, or a series of 3doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month to about 2 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose. In another embodiment, said multiple dose schedule consistsof a series of 3 doses separated by an interval of about 1 monthfollowed by a fourth dose about 10 months to about 13 months after thefirst dose, or a series of 3 doses separated by an interval of about 2months followed by a fourth dose about 10 months to about 13 monthsafter the first dose.

In an embodiment, the multiple dose schedule consists of at least onedose (e.g., 1, 2 or 3 doses) in the first year of age followed by atleast one toddler dose.

In an embodiment, the multiple dose schedule consists of a series of 2or 3 doses separated by an interval of about 1 month to about 2 months(for example 28-56 days between doses), starting at 2 months of age, andfollowed by a toddler dose at 12-18 months of age. In an embodiment,said multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 to 2 months (for example 28-56 days betweendoses), starting at 2 months of age, and followed by a toddler dose at12-15 months of age. In another embodiment, said multiple dose scheduleconsists of a series of 2 doses separated by an interval of about 2months, starting at 2 months of age, and followed by a toddler dose at12-18 months of age.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine at 2, 4, 6, and 12-15 months of age.

In an embodiment, a prime dose is given at day 0 and one or more boostsare given at intervals that range from about 2 to about 24 weeks,preferably with a dosing interval of 4-8 weeks.

In an embodiment, a prime dose is given at day 0 and a boost is givenabout 3 months later.

10 Kit and Process

In an embodiment, the invention is directed toward a kit comprising animmunogenic composition disclosed herein and an information leaflet.

In an embodiment said information leaflet mentions the ability of thecomposition to elicit functional antibodies against S. pneumoniaeserotypes 10A and/or 39.

In an embodiment said information leaflet mentions the ability of thecomposition to elicit anti-capsular antibodies against S. pneumoniaeserotypes 10A and/or 39 at a concentration 0.35 μg/mL in a humanpopulation.

In an embodiment said information leaflet mentions the ability of thecomposition to elicit OPA titers against S. pneumoniae serotypes 10Aand/or 39 in a human population.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure        and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of said composition to elicit functional antibodies        against S. pneumoniae serotypes 10A and/or 39.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure        and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of the composition to elicit anti-capsular        antibodies against S. pneumoniae serotypes 10A and/or 39 at a        concentration ≥0.35 μg/mL in a human population.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure        and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of the composition to elicit OPA titers against S.        pneumoniae serotypes 10A and/or 39 in a human population.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure;    -   printing an information leaflet wherein said information leaflet        mentions the ability of said composition to elicit functional        antibodies against S. pneumoniae serotypes 10A and/or 39;    -   combining in the same kit said immunogenic composition and said        information leaflet.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure;    -   printing an information leaflet wherein said information leaflet        mentions the ability of the composition to elicit anti-capsular        antibodies against S. pneumoniae serotypes 10A and/or 39 at a        concentration 0.35 μg/mL in a human population;    -   combining in the same kit said immunogenic composition and said        information leaflet.

In an embodiment, the invention is directed toward a process forproducing a kit comprising an immunogenic composition and an informationleaflet, said process comprising the step of:

-   -   producing an immunogenic composition of the present disclosure;    -   printing an information leaflet wherein said information leaflet        mentions the ability of the composition to elicit OPA titers        against S. pneumoniae serotypes 10A and/or 39 in a human        population;    -   combining in the same kit said immunogenic composition and said        information leaflet.

11 Methods

In an embodiment, the invention is directed toward a method comprisingthe step of:

-   -   injecting to a subject an immunologically effective amount of        any of the immunogenic compositions defined in the present        document;    -   collecting a serum sample from said subject;    -   testing said serum sample for opsonophagocytic killing activity        against S. pneumoniae serotype 10A and/or 39 by in vitro        opsonophagocytic killing assay (OPA).

12 Particular Embodiments of the Invention

Particular embodiments of the invention are set forth in the followingnumbered paragraphs:

1. An immunogenic composition comprising at least one glycoconjugatefrom S. pneumoniae serotype 39.

2. The immunogenic composition of paragraph 1 wherein, said serotype 39glycoconjugate has a molecular weight of between 50 kDa and 30,000 kDa.

3. The immunogenic composition of any one of paragraphs 1-2 wherein,said serotype 39 glycoconjugate has a molecular weight of between 4,000kDa and 25,000 KDa.

4. The immunogenic composition of any one of paragraphs 1-3 wherein,said serotype 39 glycoconjugate comprises a saccharide which has adegree of O-acetylation of between 10 and 100%.

5. The immunogenic composition of any one of paragraphs 1-3 wherein,said serotype 39 glycoconjugate comprises a saccharide which has adegree of O-acetylation of between 0 and 50%.

6. The immunogenic composition of any one of paragraphs 1-3 wherein saidserotype 39 glycoconjugate comprises at least 0.01 O-acetyl group perpolysaccharide repeating unit of serotype 39 polysaccharide.

7. The immunogenic composition of any one of paragraphs 1-3 wherein saidserotype 39 glycoconjugate comprises less than 0.5 O-acetyl group perpolysaccharide repeating unit of serotype 39 polysaccharide.

8. The immunogenic composition of any one of paragraphs 1-7 wherein thedegree of conjugation of said serotype 39 glycoconjugate is between 2and 19.

9. The immunogenic composition of any one of paragraphs 1-8 wherein, theratio (w/w) of serotype 39 capsular saccharide to carrier protein inserotype 39 glycoconjugate is between 0.5 and 3.

10. The immunogenic composition of any one of paragraphs 1-9 whereinsaid serotype 39 glycoconjugate comprises less than about 50% of freeserotype 39 capsular saccharide compared to the total amount of serotype39 capsular saccharide.

11. The immunogenic composition of any one of paragraphs 1-10 wherein atleast 30% of the serotype 39 glycoconjugates have a Kd below or equal to0.3 in a CL-4B column.

12. The immunogenic composition of any one of paragraphs 1-11 whereinthe carrier protein of said serotype 39 glycoconjugate is selected fromthe group consisting of: DT (Diphtheria toxin), TT (tetanus toxid),CRM197, other DT mutants, PD (Haemophilus influenzae protein D), orimmunologically functional equivalents thereof.

13. The immunogenic composition of any one of paragraphs 1-11 whereinthe carrier protein of said serotype 39 glycoconjugate is CRM197.

14. The immunogenic composition of any one of paragraphs 1-11 whereinthe carrier protein of said serotype 39 glycoconjugate is TT.

15. The immunogenic composition of any one of paragraphs 1-14 whereinsaid serotype 39 glycoconjugate is prepared using reductive amination.

16. The immunogenic composition of any one of paragraphs 1-14 whereinsaid serotype 39 glycoconjugate is prepared using direct CDAP chemistry.

17. The immunogenic composition of any one of paragraphs 1-14 whereinsaid serotype 39 glycoconjugate is prepared using indirect CDAPchemistry.

18. The immunogenic composition of any one of paragraphs 1-14 whereinsaid serotype 39 glycoconjugate is prepared using direct CDI chemistry.

19. An immunogenic composition comprising at least one glycoconjugatefrom S. pneumoniae serotype 10A for use in a method for preventing,treating or ameliorating an infection, disease or condition caused by S.pneumoniae serotype 39 in a subject.

20. The immunogenic composition of any one of paragraphs 1-18, whereinsaid composition does not comprise capsular saccharide from S.pneumoniae serotype 10A.

21. The immunogenic composition of paragraph 19, wherein saidcomposition does not comprise capsular saccharide from S. pneumoniaeserotype 39.

22. The immunogenic composition of any one of paragraphs 1-21, furthercomprising at least one glycoconjugate from S. pneumoniae serotype 4.

23. The immunogenic composition of any one of paragraphs 1-22 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 6B.

24. The immunogenic composition of any one of paragraphs 1-23 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 14.

25. The immunogenic composition of any one of paragraphs 1-24 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 18C.

26. The immunogenic composition of any one of paragraphs 1-25 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 19F.

27. The immunogenic composition of any one of paragraphs 1-26 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 23F.

28. The immunogenic composition of any one of paragraphs 1-21 furthercomprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 14, 18C,19F and 23F.

29. The immunogenic composition of any one of paragraphs 1-28 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 1.

30. The immunogenic composition of any one of paragraphs 1-29 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 5.

31. The immunogenic composition of any one of paragraphs 1-30 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 7F.

32. The immunogenic composition of any one of paragraphs 1-28 furthercomprising glycoconjugates from S. pneumoniae serotypes 1, 5 and 7F.

33. The immunogenic composition of any one of paragraphs 1-32 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 6A.

34. The immunogenic composition of any one of paragraphs 1-33 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 19A.

35. The immunogenic composition of any one of paragraphs 1-32 furthercomprising glycoconjugates from S. pneumoniae serotypes 6A and 19A.

36. The immunogenic composition of any one of paragraphs 1-35 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 3.

37. The immunogenic composition of any one of paragraphs 1-36 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 15B.

38. The immunogenic composition of any one of paragraphs 1-37 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 22F.

39. The immunogenic composition of any one of paragraphs 1-38 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 33F.

40. The immunogenic composition of any one of paragraphs 1-39 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 8.

41. The immunogenic composition of any one of paragraphs 1-40 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 11A.

42. The immunogenic composition of any one of paragraphs 1-41 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 12F.

43. The immunogenic composition of any one of paragraphs 1-36 furthercomprising glycoconjugates from S. pneumoniae serotypes 22F and 33F.

44. The immunogenic composition of any one of paragraphs 1-36 furthercomprising glycoconjugates from S. pneumoniae serotypes 15B, 22F and33F.

45. The immunogenic composition of any one of paragraphs 1-39 furthercomprising glycoconjugates from S. pneumoniae serotypes 12F, 10A, 11Aand 8.

46. The immunogenic composition of any one of paragraphs 1-45 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 2.

47. The immunogenic composition of any one of paragraphs 1-46 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 17F.

48. The immunogenic composition of any one of paragraphs 1-47 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 20.

49. The immunogenic composition of any one of paragraphs 1-45 furthercomprising glycoconjugates from S. pneumoniae serotypes 2, 17F and 20.

50. The immunogenic composition of any one of paragraphs 1-49 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 15C.

51. The immunogenic composition of any one of paragraphs 1-50 furthercomprising at least one glycoconjugate from S. pneumoniae serotype 9N.

52. The immunogenic composition of any one of paragraphs 1-51 which is a1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25 or 26-valent pneumococcal conjugate composition.

53. The immunogenic composition of any one of paragraphs 1-51 which is a13, 14, 15, 16, 17, 18, 19 or 20 valent pneumococcal conjugatecomposition.

54. The immunogenic composition of any one of paragraphs 1-51 which is a16-valent pneumococcal conjugate composition.

55. The immunogenic composition of any one of paragraphs 1-51 which is a20-valent pneumococcal conjugate composition.

56. The immunogenic composition of any one of paragraphs 1-55 whereinsaid glycoconjugates are individually conjugated to CRM197.

57. The immunogenic composition of any one of paragraphs 1-55 whereinall glycoconjugates are individually conjugated to CRM197.

58. The immunogenic composition of any one of paragraphs 1-55 wherein,the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14and/or 23F are individually conjugated to PD.

59. The immunogenic composition of any one of paragraphs 25-58 whereinthe glycoconjugate from S. pneumoniae serotype 18C is conjugated to TT.

60. The immunogenic composition of any one of paragraphs 26-59 whereinthe glycoconjugate from S. pneumoniae serotype 19F is conjugated to DT.

61. The immunogenic composition of any one of paragraphs 1-60 whereinsaid glycoconjugates are prepared using direct or indirect CDAPchemistry.

62. The immunogenic composition of any one of paragraphs 1-60 whereinsaid glycoconjugates are prepared by reductive amination.

63. The immunogenic composition of any one of paragraphs 33-60 whereinsaid glycoconjugate from S. pneumoniae serotype 6A is prepared byreductive amination.

64. The immunogenic composition of any one of paragraphs 34-60 whereinsaid glycoconjugate from S. pneumoniae serotype 19A is prepared byreductive amination.

65. The immunogenic composition of any one of paragraphs 36-60 whereinsaid glycoconjugate from S. pneumoniae serotype 3 is prepared byreductive amination.

66. The immunogenic composition of any one of paragraphs 1-65 whereinsaid immunogenic composition further comprises antigens from otherpathogens.

67. The immunogenic composition of any one of paragraphs 1-66 whereinsaid immunogenic composition further comprises antigens selected from: adiphtheria toxoid (D), a tetanus toxoid (T), a pertussis antigen (P),which is typically acellular (Pa), a hepatitis B virus (HBV) surfaceantigen (HBsAg), a hepatitis A virus (HAV) antigen, a conjugatedHaemophilus influenzae type b capsular saccharide (Hib) and inactivatedpoliovirus vaccine (IPV).

68. The immunogenic composition of any one of paragraphs 1-67 whereinsaid immunogenic composition further comprises at least one adjuvant,most preferably any of the adjuvant disclosed herein.

69. The immunogenic composition of any one of paragraphs 1-68 whereinsaid immunogenic composition further comprises at least one adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide.

70. The immunogenic composition of any one of paragraphs 1-69 whereinsaid immunogenic composition comprises from 0.1 mg/mL to 1 mg/mL ofelemental aluminum in the form of aluminum phosphate as adjuvant.

71. The immunogenic composition of any one of paragraphs 1-70 which isable to elicit IgG antibodies in human, and which is capable of bindingS. pneumoniae serotypes 10A polysaccharide at a concentration of atleast 0.35 μg/ml as determined by ELISA assay.

72. The immunogenic composition of any one of paragraphs 1-71 which isable to elicit IgG antibodies in human, and which is capable of bindingS. pneumoniae serotypes 39 polysaccharide at a concentration of at least0.35 μg/ml as determined by ELISA assay.

73. The immunogenic composition of any one of paragraphs 1-72 which isable to elicit functional antibodies in human, and which is capable ofkilling S. pneumoniae serotype 10A and/or 39 as determined by in vitroopsonophagocytic assay (OPA).

74. The immunogenic composition of any one of paragraphs 1-73 which isable to elicit a titer of at least 1:8 against S. pneumoniae serotype10A in at least 50% of the subjects as determined by in vitroopsonophagocytic killing assay (OPA).

75. The immunogenic composition of any one of paragraphs 1-74 which isable to elicit a titer of at least 1:8 against S. pneumoniae serotype 39in at least 50% of the subjects as determined by in vitroopsonophagocytic killing assay (OPA).

76. The immunogenic composition of any one of paragraphs 1-75 which isable to significantly increase the proportion of responders against S.pneumoniae serotype 10A as compared to the pre-immunized population.

77. The immunogenic composition of any one of paragraphs 1-76 which isable to significantly increase the proportion of responders against S.pneumoniae serotype 39 as compared to the pre-immunized population.

78. The immunogenic composition of any one of paragraphs 1-77 which isable to significantly increase the OPA titers of human subjects againstS. pneumoniae serotype 10A as compared to the pre-immunized population.

79. The immunogenic composition of any one of paragraphs 1-78 which isable to significantly increase the OPA titers of human subjects againstS. pneumoniae serotype 39 as compared to the pre-immunized population.

80. The immunogenic composition of any one of paragraphs 1-79, for usein a method of immunizing a subject against infection by S. pneumoniaeserotype 10A.

81. The immunogenic composition of any one of paragraphs 1-79, for usein a method of immunizing a subject against infection by S. pneumoniaeserotype 39.

82. The immunogenic composition of any one of paragraphs 1-79, for usein a method of immunizing a subject against infection by S. pneumoniaeserotype 10A and 39.

83. The immunogenic composition of any one of paragraphs 1-79 for use ina method for preventing, treating or ameliorating an infection, diseaseor condition caused by S. pneumoniae serotypes 10A and/or 39 in asubject.

84. The immunogenic composition of any one of paragraphs 1-79 for use toprevent serotypes 10A and/or 39 S. pneumoniae infection in a subject.

85. The immunogenic composition of any one of paragraphs 1-79 for use ina method to protect or treat a human susceptible to S. pneumoniaeserotypes 10A and/or 39 infection, by means of administering saidimmunogenic compositions via a systemic or mucosal route.

86. A method of preventing, treating or ameliorating an infection,disease or condition associated with S. pneumoniae serotypes 10A and/or39 in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition of anyone of paragraphs 1-79.

87. A method of preventing an infection by S. pneumoniae serotypes 10Aand/or 39 in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition of anyone of paragraphs 1-79.

88. The immunogenic composition of any one of paragraphs 19-87, whereinsaid subject is a human less than 1 year of age.

89. The immunogenic composition of any one of paragraphs 19-87, whereinsaid subject is a human is a human less than 2 years of age.

90. The immunogenic composition of any one of paragraphs 19-87, whereinsaid subject is a human 50 years of age or older.

91. The immunogenic composition of any one of paragraphs 1-90 for use ina multiple dose vaccination schedule.

92. A kit comprising an immunogenic composition disclosed herein and aninformation leaflet.

93. A kit comprising an immunogenic composition of any one of paragraphs1-79 and an information leaflet.

94. The kit of paragraph 92 or 93 wherein said information leafletmentions the ability of the composition to elicit functional antibodiesagainst S. pneumoniae serotypes 10A and/or 39.

95. The kit of paragraph 92 or 93 wherein said information leafletmentions the ability of the composition to elicit functional antibodiesagainst S. pneumoniae serotype 10A.

96. The kit of paragraph 92 or 93 wherein said information leafletmentions the ability of the composition to elicit anti-capsularantibodies against S. pneumoniae serotypes 10A and/or 39 at aconcentration 0.35 μg/mL in a human population.

97. The kit of paragraph 92 or 93 wherein said information leafletmentions the ability of the composition to elicit anti-capsularantibodies against S. pneumoniae serotype 10A at a concentration 0.35μg/mL in a human population.

98. The kit of any one of paragraphs 92-96 wherein said informationleaflet mentions the ability of the composition to elicit OPA titersagainst S. pneumoniae serotypes 10A and/or 39 in a human population.

99. The kit of any one of paragraphs 92-96 wherein said informationleaflet mentions the ability of the composition to elicit OPA titersagainst S. pneumoniae serotypes 10A in a human population.

100. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of of any one of paragraphs        1-79; and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of said composition to elicit functional antibodies        against S. pneumoniae serotypes 10A and/or 39.

101. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of any one of paragraphs        1-79; and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of the composition to elicit anti-capsular        antibodies against S. pneumoniae serotypes 10A and/or 39 at a        concentration ≥0.35 μg/mL in a human population.

102. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of any one of paragraphs        1-79; and    -   combining in the same kit said immunogenic composition and        information leaflet, wherein said information leaflet mentions        the ability of the composition to elicit OPA titers against S.        pneumoniae serotypes 10A and/or 39 in a human population.

103. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of any one of paragraphs        1-79;    -   printing an information leaflet wherein said information leaflet        mentions the ability of said composition to elicit functional        antibodies against S. pneumoniae serotypes 10A and/or 39; and    -   combining in the same kit said immunogenic composition and said        information leaflet.

104. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of of any one of paragraphs        1-79;    -   printing an information leaflet wherein said information leaflet        mentions the ability of the composition to elicit anti-capsular        antibodies against S. pneumoniae serotypes 10A and/or 39 at a        concentration 0.35 μg/mL in a human population; and    -   combining in the same kit said immunogenic composition and said        information leaflet.

105. A process for producing a kit comprising an immunogenic compositionand an information leaflet, said process comprising the steps of:

-   -   producing an immunogenic composition of any one of paragraphs        1-79;    -   printing an information leaflet wherein said information leaflet        mentions the ability of the composition to elicit OPA titers        against S. pneumoniae serotypes 10A and/or 39 in a human        population; and    -   combining in the same kit said immunogenic composition and said        information leaflet.

106. A method comprising the steps of:

-   -   injecting to a subject an immunologically effective amount of        the immunogenic composition defined at any one of paragraphs        1-79;    -   collecting a serum sample from said subject; and    -   testing said serum sample for opsonophagocytic killing activity        against S. pneumoniae serotype 10A and/or 39 by in vitro        opsonophagocytic killing assay (OPA).

107. A method of inducing an immune response to S. pneumoniae serotypes10A and/or 39 in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition of anyone of paragraphs 1-79.

108. Use of an immunogenic composition of any one of paragraphs 1-79 forthe manufacture of a medicament for immunizing a subject againstinfection by S. pneumoniae serotype 10A and/or 39.

109. Use of an immunogenic composition of any one of paragraphs 1-79 forthe manufacture of a medicament for preventing, treating or amelioratingan infection, disease or condition caused by S. pneumoniae serotypes 10Aand/or 39 in a subject.

110. Use of an immunogenic composition of any one of paragraphs 1-79 forthe manufacture of a medicament for preventing infection by serotypes10A and/or 39 S. pneumoniae in a subject.

As used herein, the term “about” means within a statistically meaningfulrange of a value, such as a stated concentration range, time frame,molecular weight, temperature or pH. Such a range can be within an orderof magnitude, typically within 20%, more typically within 10%, and evenmore typically within 5% or within 1% of a given value or range.Sometimes, such a range can be within the experimental error typical ofstandard methods used for the measurement and/or determination of agiven value or range. The allowable variation encompassed by the term“about” will depend upon the particular system under study, and can bereadily appreciated by one of ordinary skill in the art. Whenever arange is recited within this application, every whole number integerwithin the range is also contemplated as an embodiment of thedisclosure.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The invention is illustrated in the accompanying examples. The examplesbelow are carried out using standard techniques, which are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. The examples are illustrative, but do not limit theinvention.

EXAMPLE Example 1 Cross-Reactivity of S. pneumoniae (S. Pn.) 10AMonoclonal Antibody (mAb) with S. pneumoniae 39 Bacterial Strain

The binding specificity of S. pn. 10A mAb was evaluated by both flowcytometry and UAD assays.

For the flow cytometry study, cultured bacteria were fixed in 1%(vol/vol) paraformaldehyde, stained by S. pn. 10A mAb or control mouseIgG. The bound S. pn. 10A mAb was detected by biotinylated goatanti-mouse IgG followed by streptavidin-phycoerythrin. As shown in FIG.1, S. pn. 39 strain exhibited similar higher level of 10A mAb specificsignal compared to that of the homologous S. pn. 10A strain. There wasno significant cross-reactivity of S. pneumoniae mAb observed withstrains of all other 89 S. pneumoniae serotypes including strains of S.pneumoniae serogroup 10 (10B, 100, 10F) as well as 407 of non-S.pneumoniae microorganisms (data not shown).

The cross-reactivity of the S. pn. 10A mAb with S. pn. 39 was furtherevaluated using either crude antigens (Sheppard et al, J. Med.Microbiol. 2011, 60, 49-55.) or purified polysaccharides (Pride et al,Clin. Vaccine Immunol. 2012, 19, 1131-41) in the multiplex urinaryantigen detection (UAD) assay format.

The reactivity of S. pn. 10A mAb was presented as mean fluorescenceintensities (MFIs). The equivalent levels of the S. pn. 10A mAb specificreactivities were detected with equal amounts of crude bacterialantigens of the S. pn. 10A and S. pn. 39, as shown in FIG. 2. BaselineMFI was obtained with S. pn 11A crude bacterial lysate used as negativecontrol. Consistent with these observations, S. pn. 10A mAb reacted withS. pn. 10A and 39 CPs in UAD assay and not with other CPs of S. pn.serogroup 10: 10B, 100 and 10A (Figure-3). These data suggest sharedantigenic epitopes present in both S. pn. 10A and S. pn.39 capsularpolysaccharides (CPs), which are responsible for the reactivity with S.pn. 10A serotype specific mAb.

Example 2 S. Pn. Serotype 10A mAbs Mediate Killing of S. Pn. Serotype 39Strain

Several S. pn. serotype 10A capsular polysaccharide (CP) specificmonoclonal antibodies were tested for their capability to kill a S. pn.39 and 10A strains in OPA killing assay. S. pn. 11A strain was used asnegative control.

Results (see FIG. 4) showed that S. pn. 10A specific mAbs can mediatekilling of both S. pn. 10A and S. pn. 39 strains. There was no killingof S. pn. 11A strain observed with these mAb.

The 10A mAb OPA killing of S. pn. 10A and S. pn. 39 strains could beinhibited by the addition of both homologous and heterologous 10A and 39capsular polysaccharides respectively. Addition of serotype 10B, 100,10F, 9V and 36 CPs did not inhibit the killing (see FIGS. 5 and 6).

These results confirmed cross-reactivity results observed by FACS andUAD analysis, suggesting the presence of similar functional structuralepitopes between structures of S. pn.10A and S. pn.39 capsularpolysaccharides expressed on the bacterial cells surface of bothserotypes.

Example 3 23v S. Pn. Polysaccharide Vaccine Immune Sera can Mediate OPAKilling of Both S. Pn. 10A and 39 Bacteria

Sera from human subjects immunized with a 23v S. pn. polysaccharidevaccine (serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F), which contains S. pn.10A but not S. pn. 39 CP, were tested in S. pn. serotype 10A and S. pn.39 OPA killing assays. Data (FIG. 7) showed good correlation ofindividual sera to kill both S. pn. 10A and 39 strains with 23.6 foldincrease and 5.6 fold increase in OPA killing titer of Pn 10A and 39strain respectively.

The specificity of the functional antibodies was determined by OPAkilling inhibition studies. The human 23v S. pn. polysaccharide vaccineimmune sera from four subjects were tested in S. pn. 10A competition OPAin the presence or absence of S. pn. serogroup 10 CPs (10A, 10B, 100,and 10F), and heterologous CPs of S. pn. serotypes 9V, 36, and 39 (FIG.8). Results showed that 10A and 39 CPs inhibited OPA titers for all fourtested sera. The 10B CP was capable of inhibiting OPA killing mediatedby two out of four tested sera. Other polysaccharides did not inhibit23v CPs immune sera killing S. pn. serotype 10A strain activity.

Example 4: Preparation of Pn-39 Conjugates to CRM₁₉₇

Preparation of Isolated S. pneumoniae Serotype 39 Polysaccharide

Serotype 39 capsular polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art (see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). Streptococcus pneumoniae serotype39 were grown in a seed bottle and then transferred to a seed fermentor.Once the targeted optical density was reached, the cells weretransferred to a production fermentor. The fermentation broth wasinactivated by the addition of N-lauroyl sarcosine and purified byultrafiltration and diafiltration.

Oxidation of Isolated Streptococcus pneumoniae Serotype 39 CapsularPolysaccharide

Polysaccharide oxidation was carried out in 100 mM potassium phosphatebuffer (pH 6.0±0.2) by sequential addition of calculated amount of 500mM potassium phosphate buffer (pH 6.0) and WFI to give finalpolysaccharide concentration of 2.0 g/L. If required, the reaction pHwas adjusted to pH 6.0, approximately. After pH adjustment, the reactiontemperature was adjusted to 23±2° C. Oxidation was initiated by theaddition of approximately 0.1 molar equivalents of sodium periodate. Theoxidation reaction was performed at 23±2° C. during 16 hrs,approximately.

Concentration and diafiltration of the activated polysaccharide wascarried out using 10K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 20-fold diavolumes of WFI. The purified activatedpolysaccharide was then stored at 5±3° C. The purified activatedsaccharide is characterized inter alia by (i) saccharide concentrationby colorimetric assay; (ii) aldehyde concentration by colorimetricassay; (iii) Degree of Oxidation and (iv) Molecular Weight by SEC-MALLS

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the do/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, example by using Anthrone method. By the Anthrone mthod, thepolysaccharide is first broken down to monosaccharides by the action ofsulfuric acid and heat. The Anthrone reagent reacts with the hexoses toform a yellow-green colored complex whose absorbance is readspectrophotometrically at 625 nm. Within the range of the assay, theabsorbance is directly proportional to the amount of hexose present.

The moles of aldehyde also is determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

The conjugation process consists of the following steps:

a) Compounding with sucrose excipient and lyophilization

b) Reconstitution of the lyophilized activated polysaccharide and CRM₁₉₇

c) Conjugation of activated polysaccharide to CRM₁₉₇ and capping

d) Purification of the conjugate

a) Compounding with Sucrose excipient, and Lyophilization

The activated polysaccharide was compounded with sucrose to a ratio of25 grams of sucrose per gram of activated polysaccharide. The bottle ofcompounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20±5° C. Calculated amount of CRM₁₉₇ protein was shell-frozen andlyophilized separately. Lyophilized CRM₁₉₇ was stored at −20±5° C.

b) Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous DMSO was added to lyophilized CRM₁₉₇ forreconstitution.

c) Conjugation and Capping

Reconstituted activated polysaccharide was combined with reconstitutedCRM₁₉₇ in the reaction vessel, followed by mixing thoroughly to obtain aclear solution before initiating the conjugation with sodiumcyanoborohydride. The final polysaccharide concentration in reactionsolution is approximately 1 g/L. Conjugation was initiated by adding1.0-1.5 MEq of sodium cyanoborohydride to the reaction mixture and wasincubated at 23±2° C. for 40-48 hrs. Conjugation reaction was terminatedby adding 2 MEq of sodium borohydride (NaBH₄) to cap unreactedaldehydes. This capping reaction continued at 23±2° C. for 3±1 hrs.

d) Purification of the Conjugate

The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration. The diluted conjugate solution was passed through a 5 μmfilter and diafiltration was performed using 5 mM succinate-0.9% saline(pH 6.0) as the medium. After the diafiltration was completed, theconjugate retentate was transferred through a 0.22 μm filter.

The conjugate was diluted further with 5 mM succinate/0.9% saline (pH6), to a target saccharide concentration of approximately 0.5 mg/mL.Final 0.22 μm filtration step was completed to obtain the immunogenicconjugate. The data from the conjugates, using polysaccharides (MW range50-100 kDa) are summarized in Table 1. All the conjugates meet therequired quality attributes, including MW, SPRatio, Free Protein,Modified Lysines and Free Saccharide levels.

TABLE 1 MW50A MW50B MW75 MW100 Activation Polysaccahride MW (kDa) 12031203 1203 1203 Degree of Oxidation (DO) 11.9 11.9 19.4 23.7 ActivatedPolysaccharide 52 52 73 102 MW (kDa) Conjugation Saccharide/ProteinRatio 0.76 0.67 0.59 0.48 MW (kDa) 4111 8778 7664 15080 % FreeSaccharide 9.7 8 12.6 17.3 Free Protein <1% <1% <1% <1% Modified Lysines(AAA) 5.3 4.1 3.1 3.3

Example 5: Preparation of Pn-39 Conjugates to TT

S. pneumoniae 39 polysaccharide was activated with the cyanylatingreagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) andthen directly conjugated to the tetanus toxoid (TT) protein under mildalkaline (pH=8.2) conditions as follows:

Activation of Pn 39 Polysaccharide

0.25 mL of CDAP (100 mg/mL solution in acetonitrile) was slowly added to5.35 mL of Pn 39 polysaccharide solution (3.74 mg/mL in 0.9% NaCl). Thereaction mixture was vortexed for 5 sec. and incubated for 30 sec at25±5° C. After the incubation, 1.0 mL of 0.2 M triethylamine (TEA) wasadded and the reaction mixture was incubated for 2-2.5 min at 25±5° C.

Conjugation of Activated Polysaccharide to Tetanus Toxoid (TT)

8.3 mL of TT (3 mg/mL in 250 mM HEPES, 0.9% NaCl, pH 8.2.TT carrierprotein) was added to the activated Pn 39 polysaccharide immediatelyafter activation, using a 25 mL sterile serological pipet. Conjugationmixture was incubated at 25±5° C. for 16 hours and diafiltered against500 mL of 0.9% NaCl using a PBMK-300K Millipore membrane. Purifiedconjugates were characterized for polysaccharide and proteinconcentrations, PS:TT ratio, free saccharide and Mw (see results intable below).

Conjugate Ratio PS:TT (w:w) Free Saccharide (%) MW (kDa) Pn 39-TT 0.7:17.0 21630

Example 6: S. Pn. 39 Conjugate Hyperimmune Sera are Functional AgainstS. Pn 10A Bacteria in Killing OPA

S. pn. 39 capsular polysaccharide was conjugated to two proteincarriers, tetanus toxoid (TT) and CRM₁₉₇. S.pn. 39-TT conjugate wasprepared by CDAP chemistry (see example 5) and two S. pn. 39-CRM₁₉₇conjugates were prepared by conjugation of S. pn.39 CP activated at twoactivation levels using reductive amination chemistry (see example 4).NZW rabbits were immunized at wk. 0 and 2 with 2.2 μg of S. pn. 39conjugate+0.125 mg of AlPO4.

Sera were collected at week 0 and 4 and analyzed in OPA assays for theircapability to kill S. pn 39 and 10A bacteria.

Results (see FIGS. 9-10) showed that regardless of the protein carrier,activation level or conjugation chemistry used, S. pn. 39 CP conjugatesinduced antibodies are capable to kill both S. pn 39 and S. pn 10Abacteria.

These data demonstrate that in addition to homologous bacterial serotypestrain, S. pn. 39 CP conjugate vaccine is also able to induce functionalantibodies against heterologous S.pn 10A bacteria.

Example 7: S. pn. 10A CP Conjugate Hyperimmune Sera are FunctionalAgainst S. pn 39 Bacteria in Killing OPA

Rabbit antibodies generated by vaccination with the monovalent S. pn.10A CP-CRM conjugate (see WO2015110941) and multivalent 20V PnC vaccine(formulation of twenty different S. pn. conjugates which contained S.pn. 10A CP conjugate but not S. pn. 39 CP conjugate; see WO2015110941)were able to kill both S. pn. 10A and 39 bacterial strains in OPA assays(FIGS. 12 and 13).

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are herebyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. An immunogenic composition comprising at least one glycoconjugatefrom S. pneumoniae serotype
 39. 2. The immunogenic composition of claim1 wherein, said serotype 39 glycoconjugate has a molecular weight ofbetween 50 kDa and 30,000 kDa.
 3. The immunogenic composition of any oneof claims 1-2 wherein, said serotype 39 glycoconjugate comprises asaccharide which has a degree of O-acetylation of between 10 and 100%.4. The immunogenic composition of any one of claims 1-2 wherein saidserotype 39 glycoconjugate comprises at least 0.01 O-acetyl group perpolysaccharide repeating unit of serotype 39 polysaccharide.
 5. Theimmunogenic composition of any one of claims 1-4 wherein the degree ofconjugation of said serotype 39 glycoconjugate is between 2 and
 19. 6.The immunogenic composition of any one of claims 1-5 wherein, the ratio(w/w) of serotype 39 capsular saccharide to carrier protein in serotype39 glycoconjugate is between 0.5 and
 3. 7. The immunogenic compositionof any one of claims 1-6 wherein at least 30% of the serotype 39glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. 8.The immunogenic composition of any one of claims 1-7 wherein the carrierprotein of said serotype 39 glycoconjugate is selected from the groupconsisting of: DT (Diphtheria toxin), TT (tetanus toxid), CRM₁₉₇, otherDT mutants, PD (Haemophilus influenzae protein D), or immunologicallyfunctional equivalents thereof.
 9. An immunogenic composition comprisingat least one glycoconjugate from S. pneumoniae serotype 10A for use in amethod for preventing, treating or ameliorating an infection, disease orcondition caused by S. pneumoniae serotype 39 in a subject, wherein saidcomposition does not comprise capsular saccharide from S. pneumoniaeserotype
 39. 10. The immunogenic composition of any one of claims 1-9further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B,14, 18C, 19F and 23F.
 11. The immunogenic composition of any one ofclaims 1-10 further comprising at least one glycoconjugate from S.pneumoniae serotype 1, 5 and 7F.
 12. The immunogenic composition of anyone of claims 1-11 further comprising glycoconjugates from S. pneumoniaeserotypes 3, 6A and 19A.
 13. The immunogenic composition of any one ofclaims 1-12 further comprising glycoconjugates from S. pneumoniaeserotypes 22F and 33F.
 14. The immunogenic composition of any one ofclaims 1-13 further comprising glycoconjugates from S. pneumoniaeserotypes 8, 10A, 11A, 12F, and 15B.
 15. The immunogenic composition ofany one of claims 1-14 which is a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26-valentpneumococcal conjugate composition.
 16. The immunogenic composition ofany one of claims 1-15 wherein said immunogenic composition furthercomprises at least one adjuvant.
 17. The immunogenic composition of anyone of claims 1-16 which is able to elicit IgG antibodies in human, andwhich is capable of binding S. pneumoniae serotypes 10A and/or 39polysaccharide at a concentration of at least 0.35 μg/ml as determinedby ELISA assay.
 18. The immunogenic composition of any one of claims1-17 which is able to elicit functional antibodies in human, and whichis capable of killing S. pneumoniae serotype 10A and/or 39 as determinedby in vitro opsonophagocytic assay (OPA).
 19. The immunogeniccomposition of any one of claims 1-18 which is able to elicit a titer ofat least 1:8 against S. pneumoniae serotype 10A and/or 39 in at least50% of the subjects as determined by in vitro opsonophagocytic killingassay (OPA).
 20. The immunogenic composition of any one of claims 1-19which is able to significantly increase the OPA titers of human subjectsagainst S. pneumoniae serotype 10A and/or 39 as compared to thepre-immunized population.
 21. The immunogenic composition of any one ofclaims 1-20, for use in a method of immunizing a subject againstinfection by S. pneumoniae serotype 10A and/or
 39. 22. The immunogeniccomposition of any one of claims 1-20 for use in a method forpreventing, treating or ameliorating an infection, disease or conditioncaused by S. pneumoniae serotypes 10A and/or 39 in a subject.
 23. A kitcomprising an immunogenic composition of any one of claims 1-20 and aninformation leaflet, wherein said information leaflet mentions theability of the composition to elicit functional antibodies against S.pneumoniae serotypes 10A and/or
 39. 24. A method comprising the stepsof: injecting to a subject an immunologically effective amount of theimmunogenic composition defined at any one of claims 1-20; collecting aserum sample from said subject; and testing said serum sample foropsonophagocytic killing activity against S. pneumoniae serotype 10Aand/or 39 by in vitro opsonophagocytic killing assay (OPA).